Heterogeneous membrane electrodes

ABSTRACT

The present invention relates to planar electrochemical sensors with membrane coatings used to perform chemical analyses. The object of this invention is to provide unit-use disposable sensors of very simple and inexpensive construction, preferably with only a single membrane coating on an electrode. The invented devices are potentiometric salt-bridge reference electrodes and dissolved gas sensors constructed with a heterogeneous membrane coating of a conductor. The heterogeneous membrane, which is an intimate admixture of a hydrophobic and a hydrophilic compartment, concurrently supports constrained transport of non-volatile species through its hydrophilic compartment and rapid gas and water vapor transport through its hydrophobic compartment.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 10/856,929, filed Jun. 6, 2004, which is a continuation in partto U.S. patent application Ser. No 10/307,481 filed Dec. 2, 2002 nowU.S. Pat. No. 7,094,330, issued Aug. 22, 2006, which is incorporatedinto the present application in its entirety.

FIELD OF THE INVENTION

The invention relates to diagnostic devices comprising electrochemicalsensors for the analysis of aqueous solutions including clinicalsamples. In particular, the invention relates to the construction ofunit-use indicator and reference electrodes for such devices.

BACKGROUND OF THE INVENTION

Prior-art electrochemical sensors typically consist of anelectrochemical cell with two, sometimes three electrodes. The firstelectrode is responsive to a chemical species in the test solution andis called the indicator electrode. The second electrode called thereference electrode is typically non-responsive to changes in thecomposition of the test solution. In polarography a third,current-injecting counter electrode is sometimes used.

As is appreciated by those in the art, the performance of anelectrochemical sensor as part of a chemistry analyzer for quantitativemeasurement of chemicals in aqueous solutions is determined by itsdose-response curve. For a linear sensor this can be uniquely determinedby two coefficients: a slope and an intercept. For a dose-response curvethat is non-linear, three or more coefficients may be required. As isalso known in the art, a sensor's coefficients vary over time if it isused more than once. The coefficients also vary from sensor to sensorbecause no two sensors can be manufactured identically. Therefore, acalibration is generally required to uniquely determine a sensor'sdose-response curve. In an automated chemistry analyzer the calibrationis provided by fluidic elements (calibration fluids, pumps, valves,conduits etc.) contained within the analyzer. If a sensor is deployed asa reusable device it is often the case that the chemistry analyzer'scalibration fluidics provides for at least two calibration points and awash solution. This is because slope and intercept of the dose-responsecurve can change through repeated uses. For a unit-use device nocalibration would be required if the slope and intercept weresufficiently reproducible from sensor to sensor during manufacture andstorage. A single calibrator would be required if either one of thecoefficients was reproducible, the other not, and two calibrators ifneither coefficient was reproducible (more calibrators could be requiredfor devices with non-linear dose-response curves).

Often the goal of a manufacturer of chemistry analyzers is to producesensors sufficiently cheaply so that they can be deployed as unit-usedevices, thus eliminating or simplifying the chemistry analyzer's oftenvery complex fluidics required for the washing and calibrating ofmultiple-use sensors. To this end, manufacturers have investigatedplanar technologies for low cost sensor manufacture. Such technologiesalso purport to provide appropriate control of the materials ofconstruction and manufacturing processes to achieve device-to-devicereproducibility in high volume production.

Sensors made by planar technology have included both thick-film andthin-film micro-fabrication technologies. Thick film processed devicessuch as plastic diagnostic strips are disclosed in U.S. Pat. No.5,727,548 for example. Devices made by planar technology also includethick film processed planar substrates as in hybrid circuit or printedcircuit manufacture. U.S. Pat. Nos. 4,133,735, 4,225,410 for exampledisclose devices with electrodes made by thick film fabricationprocesses such as plating, screen-printing, dispensing and the like.

Micro-fabrication technology with its proven superior dimensionalcontrol also has been used to make devices for unit-use applications.Micro-fabrication technology employs wafer-level processes such asphotolithography on silicon wafers. U.S. Pat. Nos. 4,062,750 4,933,048and 5,063,081 disclose devices containing electrodes made by thin-filmmicro-fabrication processes on silicon substrates.

Regardless of which of the above variants of planar technology is beingused, planar devices of the prior art have been complex to manufactureand are therefore still expensive.

To better appreciate the complexity of prior-art planar sensors,consider their typical components of construction. A planarelectrochemical sensor of the prior art is a device consisting of one ormore metal conductor elements on a planar insulating substrate. Oneregion of the metal conductor element is provided for connection to anexternal measuring circuit. A planar electrode is formed in anotherregion of the metal conductor element. The planar electrode of such aprior-art electrochemical sensor consists typically of one or moreadditional metal layers (or other electrical conductors such asgraphite) and insoluble metal salt over-layers over-coating the metalconductor element. Planar electrodes are typically then coated withseveral additional functional layers as outlined below.

The planar electrode of the planar sensor is typically coated by anintegral electrolyte medium. This integral electrolyte may be a liquidaqueous solution or, more commonly, a solid hydrophilic layer such as agel material that acts like an aqueous electrolyte. In use of the planarsensor, the planar electrode region and its integral electrolyteover-layer is immersed in an aqueous solution to be tested. Chemicalspecies from the test solution permeate into the integral electrolytelayer, dissolve and often react with other reagents contained within theintegral electrolyte layer. Components of the integral electrolyte layerundergo electrochemical reaction at the electrode surface generating acurrent or a voltage. When the measured current or voltage of the sensoris selectively proportional to the concentration of a species in thetest solution that is transported from the test solution into the sensorthere is the basis for an indicator electrode for that species. If thevoltage is independent of test solution composition there is the basisfor a reference electrode. In prior-art electrochemical sensors it isgenerally required that chemical reagents within the integralelectrolyte layer be at constant concentrations during the time of themeasurement.

It is generally required that chemicals contained within the testsolution that are deleterious to the sensor reactions be rejected fromthe integral electrolyte layer. As is known in the art such contaminantsmay affect chemical reactions within the integral electrolyte layer, orthey may themselves be electro-active and cause a voltage or currentthat interferes with the measured voltage or current due to the speciesbeing analyzed. Retention of reagent chemical and rejection ofcontaminants is achieved by interposing one or more materials betweenthe integral electrolyte and the test solution. Transport of the sensedspecies from the test solution into the integral electrolyte layer takesplace by selective diffusion through the interposed materials. In manycases of prior-art planar sensors it is also necessary to interpose anadditional semi-permeable layer between the electrode and the integralelectrolyte layer. The purpose of this electrode-modifying layer is toallow transport of the chemicals of the sensor reaction while rejectingelectroactive interferents or species that poison the electrode.

In summary, as described above, planar electrochemical sensors of theprior art including the prior-art reference electrodes, enzymeelectrodes and gas sensing electrodes generally consist of numerouselements. The resulting devices are complex and costly to manufacture.To further illustrate their complexity, the devices of the prior art ineach of the above categories addressed by the current invention aredescribed in more detail in the following sections.

Potentiometric Salt-Bridge Reference Electrode Prior Art

Salt-bridge reference electrodes of the prior art consists of anelectrode, usually silver with a silver chloride over-layer which iscontacted by an integral reservoir of a concentrated aqueous solution ofa salt with equi-mobile ions, typically potassium chloride. Theelectrolyte reservoir contacts the test solution through aconstrained-flow liquid junction, which is typically a micro-porouselement. The integral aqueous electrolyte reservoir and the junctiontogether comprise a salt bridge. An ideal salt-bridge referenceelectrode of this design has an essentially constant electrode potentialand essentially zero response slope for the duration of its use. As isknown in the art of reference electrodes, the total electrode potentialis the sum of the potential difference between the electrode andintegral salt-bridge electrolyte and the liquid-junction potentialdifference which is between the salt-bridge electrolyte and the testsolution. The constant electrode potential of such prior-art referenceelectrodes is achieved firstly because the potential determiningchloride concentration of the salt-bridge electrolyte at thesilver—silver chloride electrode surface remains essentially fixed forthe duration of use. This is achieved both because the rate of chlorideefflux from the reservoir into the test solution is sufficiently smallbecause of the constrained-flow junction and because the electrolytereservoir is sufficiently large. Secondly, the response slope of suchsalt-bridge reference electrode is also small when the liquid junctionpotential difference is small as is the case when the salt-bridgeelectrolyte contains a concentrated salt with anions and cations ofnearly equal mobility, such as with the use of a concentrated potassiumchloride electrolyte.

Planar potentiometric salt-bridge reference electrodes of the prior arthave used the same approach as the classical salt-bridge referenceelectrode described above. U.S. Pat. No. 4,592,824 describes a planarsilver—silver chloride electrode on a planar silicon substrate, and asilicon cover-plate including a micro-fabricated cavity and porousregion. The cavity including the porous junction becomes the integralsalt-bridge reservoir when it is filled with concentrated potassiumchloride before use. The porous silicon element forms the region of theconstrained-flow liquid junction that contacts the test solution.Similarly, U.S. Pat. No. 4,682,602 describes a planar silver—silverchloride electrode and a cover layer defining a cavity over theelectrode. The cavity, when filled with electrolyte, becomes theintegral salt-bridge reservoir. There is a small aperture providing aflow-constraining liquid junction contact to a test solution. U.S. Pat.No. 5,385,659 describes a planar silver-silver chloride with amicro-fabricated, elongated cavity in a cover plate. When the elongatedcavity is filled with electrolyte it becomes the integral salt bridgereservoir. The flow of electrolyte out of the salt-bridge is constrainedbecause the cavity is elongated and its opening is small. These andother prior-art planar reference electrodes with integral electrolytecavities are relatively complex and costly assemblies. They must befilled with concentrated salt-bridge electrolyte before use, or, iffilled in the factory, they must be stored wet. Consequently, they areimpractical for unit-use applications.

U.S. Pat. No. 4,342,964 describes a fluidic cassette for bloodmeasurement containing a dry-stored silver—silver chloride electrodewithout an integral salt-bridge electrolyte over-layer and a spacedapart indicator electrode. In use, a calibrator solution is introducedover the pair of electrodes serving to calibrate the indicator electrodeprior to its subsequent exposure to the test solution. The calibratorsolution also fills an empty cavity region of the cassette over thesilver- silver chloride electrode and remains there to form a liquidjunction with the test solution when it is subsequently introduced intothe cassette. Thus, this patent teaches how to automatically fill areference electrode's salt-bridge reservoir without significantly addingto the complexity of the reference electrode itself, because the devicealready requires a calibrator solution and the patent teaches that thecalibrator solution can be the same as the salt-bridge filling solution.However there is added fluidic complexity and cost, and the significantlimitation on this invention is that there is no single composition ofthe calibrator solution that is satisfactory both to accuratelycalibrate the indicator electrode and provide for a low-response liquidjunction. For acceptable performance in blood it is known in the artthat the salt-bridge electrolyte should have a potassium chlorideconcentration of about 1M or even larger for the liquid junctionpotential component of the reference electrode to be acceptably smalland constant. Known calibrator solutions for blood do not provide thisconcentration

Janata in Solid State Chemical Sensors, Janata J. and Huber R. J.(eds.), Academic Press Inc., Orlando 1985, pp 101-103, describe anion-sensitive field effect reference electrode with an integralsalt-bridge reservoir formed by a hydrophilic gel layer coating theelectrode. Sinsabaugh et al. in Proceedings, Symposium onElectrochemical Sensors for Biomedical Applications, Vol. 86-14, Conan,K. N. L. (ed.), The Electrochemical Society, Pennington, N.J. 1986, pp66-73, describe a planar reference electrode consisting of asilver—silver chloride electrode over-coated by an integral salt-bridgereservoir formed by a latex membrane. In this device there are in totalthree coating steps onto the conductor element and its support. TheJanata and Sinsabaugh devices were intended for multi-use sensorapplications utilizing a calibrator solution. In a typical measurementthe reference electrode, with its salt-bridge reservoir over-layer, anda spaced-apart indicator electrode are first immersed in a calibratorsolution. The integral reservoir equilibrates to the concentration ofthe calibrator solution. When the electrode-pair is then immersed in atest solution the indicator electrode responds rapidly but, because ofits integral constrained-flow reservoir, the potential differencebetween the silver—silver chloride and the salt-bridge electrolyteover-layer responds slowly. If the reservoir thickness is sufficient(several hundred micrometers) the response is slow enough to constitutea constant potential over the time that the indicator electrode responds(approximately 10 s). During multiple uses the composition of thesalt-bridge gradually approaches the concentration of the calibrator andtest solutions in which it is immersed. These reference electrodes inmulti-use application are once again limited in utility for accurateblood measurements because the liquid junction component of thereference electrode potential is not sufficiently small or constantbecause the salt-bridge reservoir concentration is too low. Both thesepapers are silent on the use of their salt-bridge reservoirs asdry-reagent formulations in unit-use reference electrodes. Both papersare silent on the incorporation of redox chemicals into the salt-bridgereservoirs and the use of such in reference electrodes constructed withsalt-bridges coating metals. The Sinsabaugh paper is also silent on thewater vapor transport properties of their latex membrane formulation.

Because of the complexity of manufacture of reference electrodescontaining integral fluid reservoirs and because of the difficulty oftheir storage and preparation for use, a dry-reagent reference electrodeis highly desirable for unit-use applications. An integral dry-reagentsalt-bridge reservoir that contains only dry salts must first acquirewater so that the salt-bridge reservoir can ‘wet up’ to its operationalconcentration. In all of the above-mentioned prior-art devices thetransport of species through the salt-bridge reservoir and from the saltbridge to the contacting solution is through an electrolyte phase. Waterinflux for wet-up of the prior-art devices dry reagent devices isthrough the same path as potassium chloride efflux. Thus, in a devicefeaturing a constrained flow salt-bridge design with a sizeablereservoir that is required to maintain constancy of chlorideconcentration at the silver-silver chloride surface, the time for wateruptake also will be large. Also, the potassium chloride of the saltbridge electrolyte will escape from the reservoir into the solutionwhile the reservoir is acquiring water from the solution for its wet-up.Therefore, reference electrodes with dry reagent reservoirs according tothe above prior art have not been successfully deployed in unit-useapplications.

The above wet-up problem was addressed in U.S. Pat. No. 4,933,048, whichdescribes a dry-reagent salt-bridge reference electrode made by planarmicro-fabrication. In this device there is a first insulating layer on aplanar substrate that supports a conductor for connection to a measuringcircuit. A second insulating layer covers the conductor except in aregion that defines the electrode opening. There are films of silver,then silver chloride formed over the conductor in the electrode region.A solid hydrophilic material containing potassium chloride is formedover the silver chloride. This layer constitutes the integralsalt-bridge reservoir. In this device, the salt-bridge reservoir extendswell beyond the silver-silver chloride electrode edge and is furthercoated by a hydrophobic water vapor-permeable over-layer, except for aregion of the salt bridge that is far removed from the silver—silverchloride where the salt-bridge contacts the test fluid defining theliquid junction. This unit-use salt-bridge reference electrode wasdesigned to rapidly wet-up during use from its dry storage state, and toessentially retain a constantly high concentration of potassium chloridein the integral salt-bridge reservoir for a period after full wet-up andthrough the time of the measurement. These desired properties areobtained in the device of the '048 patent by providing a short diffusionpath for rapid water influx into the integral reservoir through thewater vapor-permeable over-layer and a long diffusion path for thepotassium chloride in the salt-bridge along the length of the integralreservoir. In use, the water necessary for the proper function of thesalt bridge is rapidly incorporated into the initially dry potassiumchloride layer within a few seconds by diffusion through the gaspermeable over-layer. The concentration of the internal salt-bridgeelectrolyte rapidly reaches a steady state value after a wet-up periodof a few seconds which is maintained for a period sufficient to performthe potentiometric measurement. However, this device is complex tomanufacture, consisting of five layers on top of the conductor elementand its insulating support.

U.S. Pat. No. 4,431,508 describes a graphite reference electrode with ahydrophilic coating containing a redox couple manufactured withnon-planar conventional technology.

In summary, planar reference electrodes of the prior art consist of asilver—silver chloride electrode contacting an integral salt-bridgeelectrolyte reservoir consisting of concentrated potassium chloride.These devices are either manufactured with water already incorporatedinto the salt-bridge reservoir, or, they are dry-reagent devices with agas permeable coating that facilitates water transport into the saltbridge. The salt bridge makes connection to the test solution through asmall, flow-constraining orifice or other flow limiting physicalconstriction fabricated on the device in planar technology. Theconnection of the salt bridge to the test solution is at a point removedfrom the silver-silver chloride electrode, so that an integral reservoirof electrolyte is present between the solution and the electrode.

Potentiometric Dissolved Gas Sensor Prior Art

The carbon dioxide sensor is exemplary of potentiometric gas sensors ofthe prior-art. U.S. Pat. No. 4,734,184 is one typical example from alarge literature of planar carbon dioxide sensors. In this example thedevice consists of a planar insulating substrate with two conductorelements for connection to a measuring circuit. Assembled thereon aretwo silver-silver chloride electrodes. One electrode is an internalpotentiometric reference electrode, the other electrode is furthercoated with an integral water permeable layer, then a pH sensing layerconstituting together an internal pH indicator electrode. The electrodepair is further coated with two hydrophilic matrixes containingelectrolytes, together constituting the integral internal electrolyte,and then a gas permeable membrane. Thus, the potentiometric gas sensorof this typical example requires seven coating steps onto the conductorelements and their insulating support. This device is wet-up prior touse, then immersed in a test solution containing dissolved carbondioxide. The gas diffuses through the gas permeable membrane into theintegral internal electrolyte layer where it dissolves and changes thepH of the electrolyte. The integral internal electrolyte and the twointernal electrodes are electrically isolated from the test solution bythe gas permeable membrane. The pH change of the internal electrolyte,which is related to the carbon dioxide concentration, is measured by thevoltage between the internal indicator and reference electrode.

Simplifications of the classical two-electrode carbon dioxide sensordesign have been disclosed in U.S. Pat. No. 5,496,521. This patentdescribes a carbon dioxide electrode with no internal referenceelectrode. The device comprises an indicator pH electrode an integralinternal electrolyte layer and an ionophore doped homogeneous gaspermeable over-layer. The test solution is electrically connected to theintegral internal electrolyte by the ion conduction through thehomogeneous, ionophore-doped membrane. The sensor of this constructionstill needs at least four coating layers on the conductor elements andtheir insulating substrate. Similarly, U.S. Pat. No. 5,554,272 describesa bicarbonate sensor using a homogeneous gas permeable membrane renderedion conducting by incorporation of an ionophore.

Polarographic Oxygen Sensor Prior-Art

The dissolved oxygen sensor is exemplary of polarographic gas sensors ofthe prior-art. U.S. Pat. No. 4,534,356 is one typical example from alarge literature of planar dissolved oxygen sensors. In this example,the device consists of a planar insulating substrate with two conductorelements for connection to a measuring circuit. There is a coating ofsilver, then silver chloride on one conductor element that constitutes afirst electrode, the reference electrode or anode. A coating of acatalytic metal film (gold or platinum in this example) applied over theother conductor element constitutes the second electrode, the cathode.The electrode pair is further coated with an integral electrolyte layerconsisting of a hydrophilic membrane containing dissolved salts and thena second layer which is a gas permeable membrane (Teflon in thisexample). Thus, this polarographic gas sensor consists of six coatingsteps for applying the various layers onto the conductor elements andtheir insulating support. Another typical example is U.S. Pat. No.5,246,576. In this device there are anode and cathode metal coatings ona planar substrate, with two over- layers. The first is an integralelectrolyte layer comprising a hydrophilic membrane containing salts.The second layer is formed from one or two gas permeable membranecoatings. There are a total of eight coating steps in this device. Thesedevices are wet-up prior to use so that the integral electrolyteimmersing the electrode pair already contains water and dissolved salts.In use, these devices are immersed into a test solution containingdissolved oxygen. The gas diffuses through the pas permeable membraneand then diffuses through the integral electrolyte to the cathodicelectrode surface where it is electrochemically reduced. The internalelectrolyte and the two internal electrodes are electrically isolatedfrom the test solution by the gas permeable membrane. The currentflowing between the internal anode and cathode is proportional to theoxygen concentration

Modifications to the classical polarographic oxygen sensor design aredisclosed in U.S. Pat. No. 5,514,253. This patent describes an oxygenelectrode with no internal reference anode. It consists of a cathodecoated with an integral electrolyte layer and a gas permeableover-layer. There are openings through the gas permeable over-layer sothat the integral electrolyte makes electrical contact with the externaltest solution well away from the electrode region. This configurationallows the use of an external reference electrode. However, there arestill four coating steps required in this example. U.S. Pat. No.5,078,854 discloses a polarographic oxygen electrode with an integralinternal electrolyte and a continuous (homogeneous) gas permeablemembrane over-layer. The gas permeable over-layer is renderedappropriately ion conducting by dissolving lipophilic ions into it. Aswith U.S. Pat. No. 5,514,253, this patent teaches a simplifiedpolarographic electrode with no internal reference electrode. At leastthree coating steps are required to fabricate this prior-art sensor.

It is thus an essential feature of conventional sensors of the typesdiscussed above that the integral internal electrolyte element is largeenough and sufficiently well isolated from the test solution that itbehaves as a reservoir which immobilizes the sensor's reagents withinit. In these conventional sensors the reservoir's reagent compositionthus remains essentially fixed for the duration of a measurement (exceptin the first few seconds during wet-up of dry stored devices and exceptfor the chemical reaction involving the species to be analyzed whosecompositional changes constitute the sensor reaction), and contaminantsfrom the test solution are excluded from and thus at low concentrationin this internal electrolyte reservoir. Indeed, it is most often thecase that the composition of reagents in the internal electrolytereservoir element at the electrode surface remains fixed for numerousmeasurements because these devices have been typically designed to bereusable. In these typical prior-art devices the sensor's internalelectrolyte element is completely isolated from the test solution by oneor more layers that selectively transport only the species to beanalyzed. For example, prior-art dissolved carbon dioxide and oxygensensors consist of internal electrolyte elements covering the sensors'electrodes and a selectively gas permeable, but electrolyte impermeableover-layer on top of that. In other prior-art devices, where there isdirect contact between the internal electrolyte element and the testsolution, but the internal electrolyte adjacent the electrode is farremoved from the point of contact to the test solution.

For these and other reasons prior-art planar electrochemical sensorshave required numerous electrode materials and membrane coatings toachieve the desired functionality. Prior-art planar electrochemicalsensors, therefore, are complicated and expensive to produce. Inaddition, such devices generally still also require at least a single,in-use calibration fluid step to achieve a performance equivalent tolaboratory analyzers. Even sensor designs that use micro-fabricationtechnology (U.S. Pat. Nos. 5,063,081 and 5,514,253 for example) with itshigh levels of dimensional precision have failed to achieve the standardof performance (reproducible slope and intercept of the response)required for use without a calibration step in a fluidics-free analyzer.

Manufacturers of home use glucose sensors have developed far simplerdevices that are manufactured at low cost. Such devices do not requirecalibration at the point-of-use, but they still require lot-calibrators.However, as is appreciated by those skilled in the art, these devices donot meet the performance requirements of the quantitative laboratoryanalysis and are classified as semi-quantitative. Thus there remains asignificant need to provide electrochemical sensor devices for precisequantitative analysis which are sufficiently simple in design andconstruction for use as cost-effective unit-use devices.

SUMMARY OF THE INVENTION

It is an object of this invention to provide unit-use electrochemicalsensors and their electrode components.

It is a specific object of the invention to provide unit-use salt-bridgereference electrodes and indicator electrodes manufactured assubstantially dry reagent devices, which reach their active state afterincorporation of water at the point of use.

It is an object of this invention to provide unit-use salt-bridgereference electrodes and indicator electrodes that are used with asingle calibrator solution, preferably in a device wherein theelectrodes and calibrator are all contained within a single, unit-usehousing.

It is a further object of the invention to provide salt-bridge referenceelectrodes and dissolved gas sensors each constructed with at least asingle heterogeneous membrane. The heterogeneous membrane has theproperty that it supports rapid gas and water vapor transport through ahydrophobic gas permeable compartment and constrained electrolytetransport through a hydrophilic compartment.

These and other objects are met in a device comprising an electrode foruse in an electrochemical sensor for the analysis of an aqueous sample,comprising an electric conductor; an insulating layer on the conductor,the insulating layer having a through-going aperture defining anelectrode region; and at least a heterogeneous membrane layer having gasand electrolyte conducting properties for direct contact with thesample, the heterogeneous membrane being in contact with the insulatinglayer over the electrode region and extending through the aperture intoelectrical contact with the conductor. The term ‘electrode’ as used inthis description defines an electric conductor layer covered by aninsulator layer except for an electrode region in which the conductorlayer is exposed. The electrode region can be located at an edge of theinsulator layer or within the insulator layer, in the form of athroughgoing aperture in the insulator layer.

Carbon dioxide and oxygen sensors comprising a heterogeneous membrane ofthe invention now require only a single electrode rather than theelectrode pair in the classical design for sensors of this type. Becausethe heterogeneous membranes of gas sensors of the current invention areelectrically conducting through their hydrophilic compartment anexternal reference electrode can be used with them.

The heterogeneous membrane of this invention is a formulation thatcomprises an intimate admixture of at least two compartments, ahydrophilic compartment that supports constrained transport ofelectrolyte salts and other non-volatile species and their chemicalreactions and a hydrophobic compartment that supports rapid gas andwater vapor transport. Such a heterogeneous membrane in accordance withthe invention can be used as an element of a unit-use sensor of verysimple construction.

In a first embodiment of an electrode with a heterogeneous membrane ofthe invention, the electrode comprises a single conductor element forconnection to a measuring circuit which conductor is coated by a firsthydrophilic reservoir layer which in turn is coated by a secondheterogeneous membrane layer. The heterogeneous membrane provides thedual electrolyte and gas-conducting properties required for properdevice function. In this embodiment of the invention the firsthydrophilic layer is in contact with the electrode, it is initiallysubstantially dry, and after wet-up during the use of the device, itconstitutes an internal electrolyte reservoir that contains the reagentsrequired for the electrode reaction. The heterogeneous membranepreferably supports rapid water vapor transport through its hydrophobiccompartment, to enable the wet up of the internal electrolyte reservoir.The heterogeneous membrane also enables electrical contact between theinternal electrolyte reservoir and the test solution by electrolytetransport through its hydrophilic compartment, but the permeation ratethrough the hydrophilic compartment by electrolytes and other watersoluble non-volatile species is preferably sufficiently slow that theinternal reservoir is effectively isolated from the external testsolution during the time course of the measurement.

In another embodiment, the device consists of a single conductor elementfor connection to a measuring circuit which conductor is coated with aheterogeneous membrane. The heterogeneous membrane preferably provideswithin a single element the internal electrolyte reservoir and theconstrained electrolyte transport and rapid gas transport propertiesrequired for proper device function. In this preferred embodiment theheterogeneous membrane's initially substantially dry hydrophiliccompartment, when wet up during use of the device, serves as theinternal reagent reservoir. The heterogeneous membrane's hydrophobiccompartment provides for rapid water vapor transport to wet-up thehydrophilic compartment up to the electrode surface.

By contrast with the design of conventional electrodes of the prior art,in electrodes of the current invention it is not necessary to completelyisolate the electrode's internal electrolyte reservoir from the testsolution. In preferred embodiments, the reagent composition of thehydrophilic compartment of the heterogeneous membrane, (or of theoptional additional internal reservoir in close proximity to theelectrode surface) actually can change over time during the operation ofthe device. For example, reagents may diffuse out of the heterogeneousmembrane into the test solution or contaminants permeate into themembrane from the test solution. In devices of the invention it issufficient only that the transport of reagents or contaminants throughthe membrane be sufficiently constrained that, after wet up, theinternal reservoir's composition changes only slowly and it thenfunctions as if it was effectively isolated. Surprisingly, even thoughnumerous elements that are typically necessary to be present inprior-art devices have been omitted from the simplified devices of thisinvention, the important characteristics defining quantitative sensingperformance are retained: the invented electrodes exhibit fast wet-up(important when the device is stored dry prior to use), at leastreproducible response intercepts if they are polarographic devices andat least reproducible response slopes if they are potentiometricdevices, and the devices exhibit freedom from interferences. Thus thesevery simple devices of the invention can be incorporated into ananalyzer requiring only a single in-use calibration fluid.

This invention teaches compositions of heterogeneous membranes andmethods of measurement using electrodes incorporating heterogeneousmembranes that can tolerate some loss of their reagents into the testsolution or acquire some contaminants from the test solution during use.Specifically this invention teaches the range of desirable transportproperties of heterogeneous membranes to achieve electrodes usable inaccurate and quantitative electrochemical measurements. It is desiredthat the membrane's diffusion coefficient of water vapor (and carbondioxide or oxygen for the respective gas sensors) should be at least 10times faster than the constrained diffusion of aqueous electrolytes andother water soluble species, and preferably greater than 50 timesfaster. More specifically it is preferred that gas and water vapordiffusion occurs at greater than 1×10⁻⁶ cm² sec⁻¹ and electrolyte saltdiffusion at less than 1×10⁻⁷ cm² sec⁻¹.

This invention teaches heterogeneous membranes formulated using gas andwater vapor permeable polymers such as polydimethylsiloxane, acrylatedsiloxanes, polyurethanes and the like, in intimate admixture with aninterpenetrating hydrophilic compartment typically comprisinghydrophilic polymers, electrolyte salts and other reagents. The intimateadmixture of the resultant heterogeneous membrane provides a rapid gasand water vapor transport path through the hydrophobic compartment and atortuous transport path for electrolyte salts through the hydrophiliccompartment.

Preferred heterogeneous membranes including an intimate admixture ofhydrophobic and hydrophilic compartments achieve the necessaryconstrained electrolyte transport when they have less than 5% by volumeof the hydrophilic compartment.

A preferred embodiment of a salt bridge reference electrode comprises aheterogeneous membrane of the invention having an internal reservoirincluding at least a dry redox salt but optionally other additionalsalts which together form an approximately equi-transferrent electrolytein the reservoir when it is wet up.

A preferred embodiment of a potentiometric carbon dioxide electrodeincludes a heterogeneous membrane in accordance with the invention andan internal reservoir containing at least dry bicarbonate salt and a pHsensitive redox salt but optionally also carbonic anhydrase, with thebicarbonate at a dry loading level so that the reservoir achieves abicarbonate concentration larger than 25 mM but less than 800 mM afterit wets up.

A preferred embodiment of a polarographic oxygen electrode in accordancewith the invention comprises a heterogeneous membrane of the inventionwhose hydrophobic compartment has an oxygen permeability less than about6×10⁻¹³ mole cm⁻¹ s⁻¹ atm⁻¹.

To better achieve the desired transport properties of the heterogeneousmembrane this disclosure teaches membranes cast from emulsions in whichone of the constituent components is cross-linked to further depress thesalt diffusion coefficient through the hydrophilic compartment. This canbe achieved in one of two ways. This disclosure shows that the saltdiffusion coefficient and the diffusion coefficient of othernon-volatile species through the hydrophilic compartment of aheterogeneous membrane can be engineered to be sufficiently low when themembrane's hydrophilic compartment comprises a polymer withphoto-reactive pendant groups which can cause cross-linking of thehydrophilic polymer upon photo-irradiation of the cast membrane. In analternative approach the membrane's hydrophobic compartment can becross-linked by photo-irradiation of the cast membrane when thehydrophobic compartment contains photo-cross-linking entities. Stillanother approach is to cross-link both the hydrophilic and thehydrophobic compartment. The desired result in all cases is that thehydrophilic compartment is rapidly wet up by water vapor transportthrough the hydrophobic compartment, thus achieving the required watercontent in the internal reservoir for proper electrode function, but issufficiently constrained from swelling by the above recitedcross-linking that it retains a low salt diffusion coefficient.

The invention teaches methods of preparation of heterogeneous membranesfrom oil-in-water emulsions,

This invention teaches the fabrication of an electrode comprising aheterogeneous membrane in which the membrane material in a fluid isdeposited onto a planar substrate using a micro-dispensing method.

A preferred and surprisingly simple device and its manufacturing processresults when the heterogeneous membranes of this invention arefabricated by micro-dispensing of a casting fluid containing membranecomponents onto low cost smart card-type electrode modules (as disclosedin U.S. Pat. Publ. No. 2002-0179444-A1 and in co-pending patentapplication U.S. patent application Ser. No. 10/307,481). Thesesubstrates are laminations of gold-coated copper with epoxy foils, theepoxy foil being die-cut with through-going holes at the electrodelocations, heterogeneous membranes being micro-dispensed into the epoxyholes of the electrode module. The modules' electrode surface materialis gold. Because electrode modules are supplied on a web as a 35 mmstrip, a printing process in which the membranes are dispensed onto themodules while still on the web is particularly advantageous, beingrapid, simple and low cost. Multiple different membranes, includingheterogeneous membranes for reference electrodes, carbon dioxide andoxygen sensors of this invention as well as other membrane types such asthose for ion selective electrodes and enzyme sensors, can bemicro-dispensed onto a module comprising multiple electrode locations tofabricate a low cost sensor array.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be further described by way of example only andwith reference to the attached drawings, wherein

FIG. 1A is a cross-section through a preferred embodiment of anelectrode in accordance with the invention, including a heterogeneousmembrane coating of an electrode of a laminated foil electrode module;

FIGS. B is a cross-section through an embodiment of an electrode inaccordance with the invention, including a heterogeneous membranecoating of an electrode formed on an insulating substrate;

FIG. 2A is a cross-section through a prior-art planar potentiometricdissolved carbon dioxide electrode;

FIG. 2B is a horizontal cross-section of an embodiment of apotentiometric dissolved carbon dioxide electrode according to thisinvention;

FIG. 3 is a graph of simulation data of carbon dioxide electrodes withheterogeneous membranes: electrode voltage versus time of threemembranes having hydrophilic compartments with different salt diffusioncoefficients A: 1×10⁻⁷ cm² s⁻¹, B; 3×10⁻⁷ cm² s⁻¹, C: 1×10⁻⁶ cm² s⁻¹;

FIG. 4 is a graph of simulation data of carbon dioxide electrodes withheterogeneous membranes: electrode slope for different bicarbonate saltloading of the internal reservoir;

FIG. 5 is a graph of simulation data of carbon dioxide electrodes withheterogeneous membranes: bicarbonate interference data A: sample withnormal bicarbonate B: sample with high bicarbonate C: sample with lowbicarbonate concentration;

FIG. 6A is a cross-section through a prior-art planar polarographicoxygen sensor; and

FIG. 6B is a cross-section through an embodiment of a planarpolarographic oxygen sensor according to this invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Heterogeneous MembraneElectrodes

The heterogeneous membranes according to this invention are materialsconsisting of an intimate admixture of two components. The first is ahydrophobic gas permeable material component, the second is ahydrophilic electrolyte conducting component. The heterogeneous membranecomprises these two components as physically separate compartmentswithin the membrane. The intimately admixed hydrophobic and hydrophiliccompartments comprising the membrane are a dispersion ofinterpenetrating regions of micron or sub-micron size of each component,the resulting membrane material having interpenetrating networks of thetwo compartments. In a preferred composition, the hydrophobic componentis present in large excess by volume over the hydrophilic component. Thepreferred transport property of the heterogeneous membrane of theinvention is that the membrane diffusion coefficient for particulargases through the hydrophobic compartment (water vapor for wet-up of allsensor types, oxygen or carbon dioxide for gas sensor membranes) issignificantly larger than the membrane diffusion coefficient of speciesdissolved in the water (ions and neutral non-volatile molecules)contained within the hydrophilic compartment. We have found that sensorscan be made with adequate performance attributes when the ratio of thesediffusion coefficients is about 10, but preferably the ratio should beat least 50 and better still greater than 100.

It is generally the case that prior to incorporation of water into a dryreagent electrochemical sensor such as the ones of this invention 1. Thedevice exhibits significant noise. Absent water, the bulk membranecomponents of the device are not yet sufficiently ion conducting, andtheir electrical resistance is large. 2. The electrode potentials andresponse slopes of potentiometric electrodes are erratic and varyrapidly over time. Prior to wet-up, electrochemical reactions atelectrode interfaces are slow and the electrode potential is said not tobe well poised. 3. Polarographic devices exhibit low electrode currentand large capacitive transient currents prior to wet-up. Consequentlythere is an initial time period in which a dry reagent electrochemicalsensor should be immersed in an aqueous solution during which timeperiod the device absorbs water prior to achieving its functioning stateas a sensor. This is called the wet-up time.

Wet-up of heterogeneous membranes of this invention is by waterdiffusion as vapor through the gas permeable hydrophobic compartment andthen by rapid partitioning from the gas permeable compartment into thehydrophilic compartment within the heterogeneous membrane. Thehydrophobic compartment preferably includes a polymer chosen for itslarge water vapor permeation rate, so that the wet-up step is fast.

Hydrophobic polymers with large water vapor transmission rates are knownin the art. Examples, which are typically elastomeric materials includepolysiloxanes, polyorganophosphazenes, poly-1-trimethyl-silyl-1-propyneand poly-4-methyl-2-pentyne, polyisoprenes, polybutadienes andpolyurethanes. The hydrophobic compartment of the membrane can be aliquid polymer comprised of non-cross-linked polymer or it can be asolid prepared from the liquid by addition of cross-linking agents. Thehydrophilic compartment of the admixture of the heterogeneous membranepreferably includes one or more of the following: emulsifiers,hydrophilic polymer binder, optional cross-linkers of the hydrophilicpolymer, electrolyte salts and other optional dissolved componentsdepending on the sensor. Hydrophilic polymers are well known in the art.Examples include polyvinylalcohols, polyhydroxymethacrylates,polyacrylamides, polysaccharides, cellulosic polymers and gelatins.Methods of cross-linking hydrophilic polymers also are well known in theart. Other optional constituents of the hydrophilic compartment includecatalysts, redox agents, buffers and surfactants that will beincorporated into the membrane upon preparation.

Heterogeneous membranes in accordance with the invention are preferablyprepared by casting from solutions and suspensions of the intimatelyadmixed membrane materials in volatilizable solvents. Membranes can becast from two types of casting fluids

-   -   1: from an aqueous casting-solution containing dissolved        hydrophilic components and the hydrophobic component either as a        dispersion of suspended micron or sub micron sized solid        particles of the hydrophobic polymer resin or as an emulsion of        suspended liquid hydrophobic polymer or monomer: a so-called        oil-in-water emulsion. The emulsion may comprise a liquid        suspension of a polymer resin dissolved in a hydrophobic solvent        or it can be a solvent-free liquid polymer or monomer. Monomers        or low molecular weight liquid precursors in the suspension can        be cross-linked into a solid hydrophobic polymer membrane upon        casting if the hydrophobic polymer contains reactive groups that        can cross-link, or by addition of appropriate cross-linking        additives to the emulsion.    -   2: from a non-aqueous casting solution containing dissolved        hydrophobic polymer and the hydrophilic component dissolved in        water in an emulsion with the non-aqueous solvent: a so-called        water-in-oil emulsion.

Casting membranes containing solid suspensions are possible, but notpreferred because they typically will form membranes with air pores. Thepreferred method of the invention uses oil in water emulsions.Siloxanes, particularly polydimethylsiloxane (PDMS) or derivatives ofPDMS comprising reactive pendant groups, polyurethanes and polyurethanederivatives, epoxies and derivatives with active pendant groups havebeen used for heterogeneous membrane preparations. These materials havebeen favored because they are widely used in industry and thence readilyavailable.

In principle, any method of deposition of a coating from a volatilizableliquid is feasible. The heterogenous membrane can be cast onto anelectrode using any of the methods known in the art such as dispensingthrough a nozzle, transferring a drop onto the electrode from a solidtip, spin coating, dip coating, spray coating, screen printing and thelike. Pin-transfer and nozzle micro-dispensing techniques are preferred.

Upon casting of the membrane from the casting fluid there results amembrane in which the intimate admixture of the hydrophilic andhydrophobic components of the casting fluid is retained during thedrying process. The intimately admixed hydrophobic and hydrophiliccompartments of the cast membrane are a dispersion of regions of micronor sub-micron size of each component. Depending on the specificconditions of membrane drying, the dispersion of the hydrophilic(hydrophobic) regions may comprise a dispersion of individual isolatedparticles, or particles that are partially or completely coalesced intocontinuous interconnected regions, in which case the two componentphases form a pair of continuous interpenetrating networks. In eitherevent the heterogeneous membrane comprises an intimate admixture of twocompartments: a first hydrophobic compartment which is a network ofinterconnected or partially interconnected channels of hydrophobicmaterial through which a gas may be transported and whose channelcross-section is preferably of the order of a few micrometers or less,and a second hydrophilic compartment which is a network ofinterconnected or partially interconnected channels of hydrophilicmaterial through which an electrolyte may be transported and whosechannel cross-section is also preferably of the order of a fewmicrometers or less.

The specific device dimensions and composition of the heterogeneousmembrane element will be different for each of the electrode typesencompassed by this invention. These will be described in more detail inthe following sections.

Devices of this invention encompass sensors that function aspotentiometric salt bridge reference electrodes, and potentiometric andpolarographic gas sensors, but the inventor clearly contemplates theextension of these design principles to other sensor types such asenzyme electrodes.

All of the various principal electrode types achievable with theheterogeneous membrane technology of the current invention are depictedin the preferred embodiment of the invention shown in FIG. 1A and analternative embodiment shown in FIG. 1B. In these figures the specificcompositions and dimensions of the elements will depend on the specificelectrode type. As will be apparent from the following detaileddescriptions of each of the different electrode types, the composition,structure and dimensions of the membranes 6A, 6B determine thefunctional properties of the respective electrode.

FIG. 1A depicts the preferred laminated foil electrode embodiment, whileFIG. 1B depicts a coated electrode on an insulating substrate. Bothfigures illustrate a pair of electrodes to show how multiple electrodescan be produced on a single foil-type electrode module or on a singleinsulating substrate. It is clearly contemplated in this invention thatthere could be numerous different combinations of electrodes on a singlemodule as determined by the test application. For example a test devicefor blood gases (pH, dissolved carbon dioxide and dissolved oxygen)would consist of an array of 4 electrodes on a module (indicatorelectrodes for pH and the two dissolved gases and a common salt-bridgereference electrode) and a fifth grounding electrode. A glucose testdevice would be an array of two electrodes on a module and so on.

The laminated foil embodiment of FIG. 1A shown in cross-section includesan electrode module with a pair of electrodes, as described in detail inU.S. Pat. Publ. Nos. 2002/0179444A1, 2003/0148530A1 and co-pending U.S.patent application U.S. patent application Ser. No. 10/307,481. Theelectrode module includes an insulator foil 2 laminated with a metalfoil formed into two elements 3A, 3B and optional adhesive 4therebetween. Apertures 5A and 5B extend through the insulator anddefine the position of the two electrodes. Coatings 6A and 6B areapplied over the apertures and extend thereinto, with overlap onto theinsulator (contacting at least the vertical wall of the insulator in theaperture or even beyond onto the planar insulator surface perimetric tothe aperture). The coatings 6A, 6B are in electrical contact with themetal foil elements at 3A, 3B.

The coated insulating substrate embodiment of an electrode module 10 isshown in cross-section in FIG. 1B including a pair of electrodes. Aplanar insulating substrate 11 supports a metal film formed into twoelements 12A, 12B coated by an insulating over-layer 13. Apertures 14Aand 14B extend through the insulating over-layer and define therespective position of the two electrodes. Coatings 15A and 15B extendinto the apertures, overlap onto the insulating over-layer, and makecontact to the conductors 12A, 12B.

There are two principal variants of the membrane configuration of thedevices of FIGS. 1A and B. In the first variant there is only a singleheterogeneous membrane overlaying the conductor. In the second variantthere is an internal hydrophilic reservoir layer coating the conductor,then a second over-layer of a heterogeneous membrane. In either variant,coatings 6A and 6B of FIGS. 1A and 15A and 15B of FIG. 1B comprise oneor more membrane elements with at least one heterogeneous membraneelement according to this invention.

Heterogeneous Membrane Transport Properties

Consideration of the membrane's transport properties is needed to betterunderstand the design rules for the selection of materials andcomposition of the heterogeneous membrane according to this invention.To model the transport properties of the heterogeneous membrane oneneeds to know the transport properties of the materials of its transportcompartments and the nature of their admixture, particularly therelative volume of the hydrophobic and hydrophilic compartments, thecharacteristic dimensions of the hydrophilic compartment's transportpaths and the tortuosity of the species transport networks created whenthe two components are intimately admixed.

The tortuosity of a membrane's transport path describes the reduced rateof species diffusion relative to diffusion through a slab of purematerial. In a heterogeneous membrane of this invention the tortuositycan be modeled by the increased path length for transport of acontinuous path or by the reduced rate of particle transport fromisolated islands within a discontinuous path. Both such models oftransport are well known in the art of membrane transport.

A heterogeneous membrane of this invention is a slab of geometric area Aand geometric thickness L and volume V=AL which comprises a volume V_(G)of a gas (water vapor) permeable polymer of the hydrophobic compartmentand V−V_(G)=V_(H) of a hydrophilic compartment.

The heterogeneous membrane has two transport paths through itsthickness. There is a first transport path for gas and water vaporthrough the hydrophobic polymer compartment. The hydrophobic polymer isa material characterized by a gas (water vapor) solubility S_(G) (S_(W))moles cm⁻³ atm.⁻¹ and a gas (water vapor) diffusion coefficient D_(G)(D_(W)) cm² sec⁻¹. When the membrane is contacted by an adjacent liquidwater phase the membrane absorbs water as vapor through the hydrophobiccompartment, coming to an equilibrium water content of S_(G)P moles ofwater per cm³ of the hydrophobic compartment where P in atmospheres isthe saturated vapor pressure of water. The hydrophobic gas/water vaportransport path is characterized by an effective area A_(G), and aneffective length L_(G). The ratio L_(G)/L>1 characterizes a longertransport path for gaseous permeant than the geometric thickness. Theratio (L_(G)/L)²=τ_(G) characterizes the tortuosity of the gas permeantpath. For a heterogeneous membrane in which the predominant volumecomponent is the hydrophobic compartment, V_(G)/V>>0.5, the tortuositywill be in the range 1<τE_(G)<2. The effective diffusion coefficient ofgas/water vapor through the gas permeable path of the heterogeneousmembrane is D_(G,M) given by D_(G,M)=D_(G)/τ_(G) where the effectivediffusion coefficient relative to the membrane is less than thediffusion coefficient in a slab of the pure hydrophobic polymer D_(G) bythe tortuosity factor τ_(G). As noted, we have preferred polysiloxanesand derivatives thereof and polyurethanes and derivatives thereof as apreferred gas permeable material because of their high water vaporpermeation rate. Published data for gas solubility and diffusioncoefficient and permeability for these polymers and others are shown inTable I. Published data for a given class of materials is quite variablebecause it depends on the degree of cross-link of the material,permeability being higher for lower cross-linked elastomers.Polydimethylsiloxane has the highest permeability and diffusioncoefficient of the common elastomeric polymers(poly-1-trimethyl-silyl-1-propyne is reported to be even higher.)

TABLE 1 S P = DS D mol. cm⁻³ mol. cm cm⁻² polymer Gas cm² sec⁻¹ atm⁻¹sec⁻¹atm⁻¹ polydimethylsiloxane H₂O   1 × 10⁻⁵ 1 × 10⁻³ 1 × 10⁻⁸ polyether-urethane H₂O 3 × 10⁻⁸  polyester-urethane H₂O 4 × 10⁻⁹ polybutadiene H₂O 2 × 10⁻⁹  polyisoprene H₂O 8 × 10⁻¹⁰polydimethylsiloxane CO₂ 1.1 × 10⁻⁵ 6 × 10⁻⁵ 7 × 10⁻¹⁰polyether-urethane CO₂ 1 × 10⁻¹⁰ polyester-urethane CO₂ 6.1 × 10⁻¹²  polybutadiene CO₂ 1.1 × 10⁻⁶ 4 × 10⁻⁵ 4.7 × 10⁻¹¹   polyisoprene CO₂ 1.3× 10⁻⁶ 4 × 10⁻⁵ 5.2 × 10⁻¹¹   polydimethylsiloxane O₂   2 × 10⁻⁵ 1.5 ×10⁻⁵   3 × 10⁻¹⁰ polyether-urethane O₂ 1 × 10⁻¹¹ polyester-urethane O₂ 4× 10⁻¹³ polybutadiene O₂ 1.5 × 10⁻⁶ 4 × 10⁻⁶ 6.5 × 10⁻¹²   polyisopreneO₂ 1.7 × 10⁻⁶ 5 × 10⁻⁶ 7.9 × 10⁻¹²  

A second transport path for electrolyte salts and non-volatile moleculesis through the hydrophilic compartment after it has wet up. Thehydrophilic compartment is characterized by a solubility of water S_(H)moles cm⁻³ atm.⁻¹. When equilibrated with water at a temperature T thereare S_(H)P moles of water per cm³ of the hydrophilic compartment where Pin atmospheres is the saturated vapor pressure of water at temperatureT. The transport path is characterized by an effective area A_(H), andan effective length L_(H). The ratio L_(H)/ L>1 characterizes a longertransport path than the geometric thickness. The ratio (L_(H)/L)²=τ_(H)characterizes the tortuosity of the hydrophilic path. When the amount ofhydrophilic component in the heterogeneous membrane is large, thehydrophilic compartment comprises continuous connected conduction pathswithin the heterogeneous membrane and τ_(H) will be on the order ofunity. When the amount of hydrophilic component in the membrane is small(V_(H)/V<<0.5), the hydrophilic compartment's paths are tortuous or evenpartially discontinuous and τ_(H) will be large, and in the limit of avery small volume fraction of hydrophilic component, τ_(H) approachesinfinity and there is no longer a continuous hydrophilic conduction paththrough the membrane.

The hydrophilic compartment is further characterized by a model ofwater-containing micro-capillary pores contained within the hydrophilicmatrix. The volume of aqueous electrolyte in the hydrophilic compartmentis V_(E) , the volume of the dry other hydrophilic compartment'sconstituents being V_(H)−V_(E). At equilibrium after wet up of themembrane, V_(E)/V_(H)=S_(H)P/0.055, assuming 0.055 moles of water occupy1 cm³. The electrolyte transport path within the hydrophilic compartmentis characterized by an effective area A_(E) and an effective lengthL_(E). The ratio L_(E)/L_(H)>1 characterizes a longer transport path forelectrolyte diffusant through the pores of the hydrophilic compartmentthan the hydrophilic compartment's path length. The ratio(L_(E)/L_(H))²=τ_(P) characterizes the tortuosity of the electrolytepores relative to the hydrophilic path. It is well known in the art ofhydrophilic polymer gels that τ_(P), the tortuosity of the electrolytepath through the pores of a hydrophilic polymer matrix, can be verylarge depending on the equilibrium water content of the matrix (alsorelated to the swelling factor). The smaller the water content thelarger the tortuosity, so that typically 1<τ_(P)<1000 when1>V_(E)/V_(H)>0.01. Consequently it is possible hydrophilic matrixeswhere the water content is of the order of a few percent of the volumeof the hydrophilic matrix and the diffusion coefficient of aqueousdiffusants in the hydrophilic matrix is up to 100 or more times lowerthan the diffusion coefficient in water. (see for example Hydrogels inMedicine and Pharmacy, CRC Press, N. A. Peppas ed., Vol 1 1986). Fordiffusion of small molecules through a hydrophilic polymer containingV_(E)/V_(H) volume fraction of water, the diffusion constant of a saltthrough the hydrophilic compartment D_(H) is less than the diffusioncoefficient in water D_(W) by a factor given by

$\begin{matrix}{\frac{D_{H}}{D_{W}} = {\frac{1}{\tau_{P}} = ^{N{({1 - \frac{V_{H}}{V_{E}}})}}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

where N is a constant close to unity (see for example H. Yasuda et al. “Permeability of Solutes through Hydrated Polymer Membranes” in DieMakromolekulare Chemie 118 (Nr. 2858), (1968) p19-35).

The constraint of water uptake and resultant swelling of the hydrophiliccompartment of the wet-up heterogeneous membrane is thus often necessaryto achieve the desired low salt diffusion coefficient, and can beachieved in one of two ways: by cross-linking of the hydrophilic matrixor by cross-linking of the hydrophobic matrix, both techniques providingthe elastic compressive forces that counteract the swelling of thehydrophilic compartment during wet up. We demonstrate both approaches inthis disclosure. The literature of hydrophilic polymers (of which thetwo above citations are typical) provides numerous examples of chemicalcross-linking methods to achieve hydrophilic polymers with differentamounts equilibrium water uptake and consequently different saltdiffusion coefficients. The literature of gas permeable hydrophobicpolymers too, contains numerous examples of their cross-linkingchemistry.

Combining the tortuosity of the electrolyte path in the hydrophilicmatrix and the tortuosity of the hydrophilic matrix path within theheterogeneous membrane gives the total tortuosity of the electrolytepath with respect to the membrane as (L_(E)/L)²=τ_(P)τ_(H)=τ_(E). Theeffective diffusion coefficient of a species dissolved in the pore waterof the hydrophilic compartment of a heterogeneous membrane is D_(E,M)given by D_(E,M)=D_(E)/τ_(E) where the effective diffusion coefficientrelative to the heterogeneous membrane is less than the diffusioncoefficient in a slab of pure aqueous electrolyte D_(E) by thetortuosity factor τ_(E).

As discussed, the transport of gas and water vapor through theheterogeneous membrane is primarily by diffusion through the gaspermeable compartment and then by partitioning from the gas permeablecompartment into the intimately admixed hydrophilic compartment withinthe membrane. The partitioning of water between the hydrophobic andhydrophilic compartments' pores can be assumed to be an equilibriumprocess when the transport of water across the hydrophobic/hydrophilicpore boundary is rapid compared to transport along the pore through thethickness of the membrane. The characteristic distance of hydrophobic tohydrophilic pore transport is on the order of the pore size of theadmixture (on the order of a few micrometers or less) which is smallcompared to the membrane thickness (on the order of 100 micrometers).When transport of water from the hydrophobic compartment to thehydrophilic compartment is slower, such as when the characteristic poresize of the heterogeneous admixture is large, an additional timeconstant is introduced to the water absorption kinetics. When the wateruptake into the hydrophilic pore is a slow process, then too there is anadditional time constant in the water absorption kinetics.

The transport of electrolyte is through the water-filled capillary poreswithin the hydrophilic compartment only.

To better understand the required range of transport properties of theheterogeneous membranes of this invention we have performed simulationsof the invented electrodes' response characteristics using a finitedifference numerical method. With this method we solved the equationsdescribing the simultaneous transport of the various species through theheterogeneous membrane. The results of this simulation are the species'concentrations (water, ions other solutes and gases) within the membraneversus position and time. These concentration values are then used tocalculate the electrical responses of electrodes using heterogeneousmembranes of this invention. These numerical simulations and the datafrom exemplar heterogeneous membrane electrodes made in accordance withthis invention are presented below to teach how to best practice theinvention.

Diffusion of Water into Heterogeneous Membranes

We have computed the wet up of heterogeneous membranes as follows: Firstwe calculate the time and position dependence of water diffusing as thevapor into the membrane through the hydrophobic compartment. Thenumerical solution of the transport equations used an initial conditionof 0.01S_(W) moles cm⁻³ of water corresponding to the initialequilibrium water content of a hydrophobic polymer with water solubilityS_(W) moles cm⁻³ atm.⁻¹ initially stored in an ambient of 0.01atmospheres of water vapor (corresponding to normal room air at 23° C.and 40% RH). The solubilities and diffusion coefficients used in thesecalculations are those shown in Table 1 for highly water vapor permeablepolymers. The amount of water in the hydrophilic compartment is obtainedby computing the equilibrium partitioning between the hydrophobic andhydrophilic compartments (assuming a value for the equilibrium wateruptake of the hydrophilic compartment, this being related to theequilibrium swell factor determined by the degree of cross-linking ofthe membrane). The amount of water versus time at the inner membranesurface at the electrode is thus obtained. The time to 95% water uptakeat the inner surface, t₉₅, is then obtained from the computed timetransient.

The results of this computation are: the wet-up time increases linearlywith the equilibrium amount of water taken up by the membrane: thewet-up time increases as the square of the membrane thickness. Thesedata can be reduced to a single equation that engineers can use tocalculate wet up time for a particular membrane formulation.

$\begin{matrix}{t_{95} = {\frac{L^{2}}{P_{W}}\left( {\frac{V_{E}}{V} + {1.2S_{W}}} \right)}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

P_(W) being the hydrophobic polymer's water vapor permeability(P_(W)=D_(W)S_(W)) in units of mole-cm/cm²-sec-atm., L being themembrane thickness in cm., S_(W) being the hydrophobic polymer's watervapor solubility in units of mole /cm³-atm.

Typical membrane compositions according to this invention have a volumefraction of the hydrophilic compartment between 1% and 5% i.e.,0.01(1%)<V_(H)/V<0.05(5%), and a water uptake into the hydrophiliccompartment of between 1% and 20% volume fraction of the hydrophiliccompartment i.e., 0.01(1%)<V_(E)/ V_(H)<0.2(20%). The total volumefraction of water in the wetted-up membrane is accordingly in the range0.0001(0.01%)<V_(E)/V<0.01(1%).

A heterogeneous membrane formulated with polydimethylsiloxane, whosewater vapor solubility is 1×10⁻³ moles cm⁻³ atm.⁻¹ and diffusioncoefficient is 1×10⁻⁵ cm² sec⁻¹, at a typical thickness of 0.005 cm.absorbing 1% water has a wet-up time of t₉₅=28 seconds calculated fromequation 2. Such a formulation will still wet up quickly (t₉₅=90 secs)even when it takes in 3% water, or if it takes in 1% water and it is0.009 cm thick.

A heterogeneous membrane formulated with a less water vapor permeablepolymer, say one whose water vapor solubility is only 1×10⁻⁴ moles cm⁻³atm.⁻¹ and diffusion coefficient is 1×10⁻⁶ cm² sec⁻¹, must be formulatedwith a smaller water absorption capacity or it must be made thinner toalso wet-up rapidly. For example, with 0.1% water uptake and 0.0028 cmthickness equation 2 predicts a wet-up time of t₉₅=88 seconds

In performance tests of experimental heterogeneous membrane electrodesdescribed below we have experimentally confirmed the finite elementsimulation's predictions of the wet-up time. We have used the aboverelations to determine the useful composition range and membranethickness for rapid wet-up, being defined as t₉₅ less than about 100seconds.

Details of Membrane Cocktail Preparation

Membrane cocktails (the formulation used for printing membranes) weregenerally formulated as oil-in-water emulsions. The general procedurefor preparation of an emulsion was as follows:

-   1. The components of the hydrophilic compartment were first    pre-mixed by dissolving them in an aqueous solution. These    components include the hydrophilic binder (polyvinyl alcohol or    another hydrophilic polymer) or an emulsifier, and salts.-   2. Next the components of the oil phase were pre-mixed. These    include the hydrophobic polymer (typically a low to medium molecular    weight polymer) and optional cross-linkers.-   3. The oil and water components were mixed to a smooth blend    avoiding foam formation. 4. The oil-water blend was emulsified as    follows    -   the best results were obtained when emulsification was performed        on ice    -   2-4 mL batches of emulsion were prepared in an 8 mL vial using        an 8 mm rotor equipped on either an IKA Ultraturrax T25 (500        watt) for viscous formulations or IKA Ultraturrrax T8 (100 watt)        blender for non-viscous formulations.    -   The actual emulsification protocol depended on the formulation,        but a typical protocol employed was one where the shear rate is        gradually increased during the emulsification process, i.e. 1-2        minutes at 6,000-8,000 rpm, 1-2 minutes at 15,000 rpm and 1-2        minutes at 24,000 rpm.    -   Best emulsification was obtained when a high viscosity aqueous        component was formulated using a relatively higher concentration        of dissolved hydrophilic polymer solids.    -   A desirable emulsion according to the above procedure achieved a        high specific surface area of about 2.5 m²/mL. This corresponds        to particle dimensions of less than 1 micrometer. Larger        particle size emulsions are not preferred because: the emulsion        isn't stable over time; the hydrophilic compartment of the cast        membrane is not sufficiently tortuous; wet-up is not uniform.-   5. Cocktails were stored in a stoppered vial (a dark vial for    photo-cross-linkable formulations) until membrane printing. Pot life    of a properly emulsified formulation is generally weeks, but new    batches were typically prepared weekly.

Details of Membrane Printing, Curing and Cross-linking

Heterogeneous membrane electrodes were fabricated on smart-card typeelectrode modules. These were designed to our specified electrodegeometries and purchased from a vendor of smart card modules. Themodules comprised an epoxy foil body approximately 1 cm×1 cm and 0.01 cmin thickness with one side laminated with a 0.0015 cm copper foil whichwas plated with gold. The metal foil had been photo-formed into 8contact pads in a geometry similar to the ISO standard for smart cardmodules. There were eight 0.7 mm diameter holes die-cut through theepoxy foil in regions above the contact metal.

The modules were used for preparation of electrodes as received from thevendor. Membranes were printed by the pin-transfer printing technique aswell as micro-dispensing from a fine nozzle. The nozzle dispensetechnique is preferred because it is more appropriate for scaling tohigh volume. In the pin transfer method a metal pin was immersed intothe print cocktail to acquire a charge of print material. The pin withprint material was then transferred to the surface of the module in theregion of a hole in the epoxy. The print charge was deposited over thehole when the pin with its print material was brought into contact withthe module surface. In the nozzle dispense technique the print cocktailwas loaded into the barrel of a syringe dispense tool. The syringe tipwas 27 to 32 gauge stainless steel. During printing the syringe tip waslocated in close proximity over the print hole in the module's epoxy anda controlled volume of fluid was dispensed into the hole by applying apressure to the fluid in the syringe barrel. Print cycle-time was under1 second. The applied pressure required to deliver a known volume offluid depended on the viscosity of the cocktail.

The wet thickness of the print was typically about 0.02 to 0.05 cm andthe diameter about 0.1 cm.

Wet printed membranes were allowed to air dry at room temperature.Membranes containing photo-cross-linkable components were thenflood-exposed to UV from a commercial high intensity UV lamp (EFOActicure A4000, set at 6 W cm⁻²). The exposure time depended on thespecific formulation and the membrane thickness but was typically a fewseconds. The dry, cured membranes were soft elastomers with a thicknessin the range 0.002 to 0.01 cm, depending on the electrode type.

For test devices we typically printed several electrodes per module witha given cocktail. Modules were stored at room temperature (20-25C) andhumidity (40-50% RH) prior to testing.

Details of Electrode Testing

Preliminary electrode evaluations were performed on modules mounted in aflow cell. Qualified membrane formulations were then tested on modulesassembled into diagnostic cards in a card reader.

For electrodes which were tested in a fluidic cell, the cell comprised afluidic chamber for introduction of aqueous fluids. The cell consistedof two spaced-apart planar surfaces, one being the electrode surface ofthe module for test, the other a slab of polycarbonate. The surfaceswere spaced apart by a silicone rubber gasket which fluidically sealedthe chamber. Fluids were introduced to the chamber through a first inletpipe and removed through a second outlet pipe each connected through thepolycarbonate slab. The contact surface of the module was contacted by asmart-card connector manufactured by Amphenol. There was a silver groundelectrode in the inlet pipe and a commercial 3M KCl silver/silverchloride reference electrode (Microelectrodes Inc.) in the outlet pipe.For potentiometric measurements each of the reference electrodes on thearray of smart-card electrodes, and the in-line commercial referenceelectrode was connected to a high impedance source follower amplifierand then to a PC through a data acquisition card. For current—voltagemeasurements a voltage was applied to the in-line silver electrode andthe electrodes on the module were connected to current to voltageconverters and then to a PC through a data acquisition card.

For electrodes tested in diagnostic cards, modules with printedelectrodes were assembled into diagnostic cards also comprising anon-board calibrator in a sealed pouch. Details of the card constructionand operation were previously disclosed in U.S. patent application Ser.No. 10/307,481. Card readers were similar to those disclosed in U.S.Pat. Publ. 2003/0148530A1.

Potentiometric Salt-Bridge Reference Electrode with HeterogeneousMembrane

The principle of operation of heterogeneous membrane salt-bridgereference electrodes of this invention is described in the relatedapplication U.S. patent application Ser. No. 10/307,481. Briefly, thehydrophilic compartment of the heterogeneous membrane referenceelectrode is loaded with an equi-transferent electrolyte (a single saltwith equi-mobile ions such as potassium chloride, potassium nitrate orsodium formate for example, or a mixture of salts exhibitingequi-transference of anions and cations) and a redox salt to provide apoised potential at the inner membrane/electrode interface. The '481patent application gave several examples of heterogeneous membranereference electrodes that were formulated from commercially availablepolydimethylsiloxane emulsions.

This disclosure supplements those data with further examples of thetechnology, particularly as we have extended it to include emulsionsthat we have prepared in our laboratory.

Reference Electrode Examples:

We have investigated several families of formulation, each familydenoted in the text below as a numbered series. Formulations are oil inwater emulsions from which membranes are cast. The oil phase containsthe components of the membrane's hydrophobic compartment, while thewater phase contains components of the membrane's hydrophiliccompartment. The formulation families are shown in the table below. Theyare arranged according to whether the membrane's hydrophobic orhydrophilic compartment are cross-linked. Preferably at least one of thecompartments is cross-linked to achieve sufficiently long-livedreference electrodes wherein the electrolyte salts of the membrane'sliquid junction do not diffuse out too quickly during use, nor docontaminants diffuse in too quickly.

TABLE 2 Reference Hydrophobic Membrane compartment Hydrophiliccompartment Formulation # Non-crosslinked Non-crosslinked I n/a n/aNon-crosslinked Crosslinked II Polydimethylsiloxane SBQ derivatizedpolyvinyl a alcohol Polydimethylsiloxane Polyvinyl alcohol with bammonium dichromate Crosslinked Non-crosslinked III Acrylated siloxanePolyvinyl alcohol a Acrylated siloxane Surfactant b Urethane acrylic — cAminosiloxane + Surfactant d acrylated siloxane Crosslinked CrosslinkedIV Fotecoat emulsion — a Acrylated siloxane SBQ derivatized polyvinyl balcohol Acrylated siloxane SBQ derivatized polyvinyl c alcohol

We have investigated hydrophilic compartments with polyvinyl alcoholbinder or no binder but comprising emulsifying surfactants only. We alsoshow examples of hydrophobic compartments comprising both siloxanes andurethanes.

Formulation IIa

-   Oil:-   1.5 g polydimethysiloxane (Aldrich, 378402, 10,000 cSt)-   0.5 g hexamethyldisiloxane (Aldrich, 205389)-   Water:-   0.06 g polyvinylalcohol (Fluka, 18-88), derivatized with    2.75%(+/−0.25%) SBQ-   1.22 g DI water-   0.2 g 0.2M potassium chloride solution

Derivatization of polyvinylalcohol by SBQ(N-methyl-4-(p-forylstyryl)pyridinium methosulfate, from EsprixTechnologies) was performed by us according to procedures described inthe literature (for example K. Ichimura, J. Polymer Sci., 22, 2817-2828,1984)

Formulation IIb

-   Oil:-   1.0 g polydimethylsiloxane (Aldrich, 378402, 10,000 cSt)-   0.35 g hexamethyldisiloxane (Aldrich, 205389)-   Water:-   0.06 g polyvinylalcohol (PolyScience, 49-88)-   0.9 g DI water-   0.48 g 0.1M ammonium dichromate solution-   50 microL 200 mM potassium chloride solution

Formulation IIIa

-   Oil:-   2.2 g 5% acrylated siloxane (Gelest, UCS-052, 150-200 cSt)-   0.06 g α-hydroxycyclohexylphenylketone (Aldrich, 405612)-   0.06 g αα-dimethyl-α-phenylacetophenone (Fluka, 38781)-   Water:-   0.1 g polyvinylalcohol (Fluka, 18-88)-   1.9 g DI water-   0.1 g 0.1M potassium ferricyanide solution-   0.1 g 0.1M potassium ferrocyanide solution

We have also made similar formulations using higher percent acrylatedsiloxanes such as 10% acrylated siloxanes (from Rhodia, Rhodosil R01194,800 cSt) sensitized with 2.5% by weight ofαα-dimethyl-α-phenylacetophenone (Fluka 38781) and 99% (acryloxypropyl)methylsiloxane, sensitized (Gelest, Zipcone UA, 100 cSt). Increasingacrylation above 10% did not improve the membrane performance butresulted in slower wet-up, and we have preferred the low acrylatedsiloxane formulations.

Formulation IIIb

-   Oil:-   1.0 g 10% acrylated siloxane (Rhodia, Rhodosil R01194, 800 cSt)-   0.025 g α-hydroxycyclohexylphenylketone (Aldrich, 405612)-   0.025 g α-dimethyl-α-phenylacetophenone (Fluka, 38781)-   Water:-   0.1 g 75EO-DMS, dimethylsiloxane-75% ethylene oxide copolymer    (Gelest, DBE-712)-   0.1 g 0.1M potassium ferricyanide solution-   0.1 g 0.1M potassium ferrocyanide solution-   20 microL 50 mM potassium chloride solution

Similar results were obtained with other emulsifying surfactants such aspluronic P123 (from BASF) and carbinol-siloxane

Formulation IIIc

-   2.0 g urethane acrylic emulsion, Joncryl U6070 (from Johnson    Polymer)-   0.05 g 0.1M potassium ferricyanide solution-   0.05 g 0.1M potassium ferrocyanide solution-   20 microL 50 mM potassium chloride solution

Formulation IIId

-   Oil:-   0.475 g 2-3% aminopropylmethylsiloxane-dimethylsiloxane copolymer    (Gelest, AMS132, 100 cSt)-   0.475 g 10% acrylated siloxane (Rhodia, Rhodosil R01194, 800 cSt)-   0.025 g α-hydroxycyclohexylphenylketone (Aldrich, 405612)-   0.025 g αα-dimethyl-α-phenylacetophenone (Fluka, 38781)-   Water:-   0.02 g Triton X100-   0.125 g 0.1M potassium ferricyanide solution-   0.125 g 0.1M potassium ferrocyanide solution-   15 microL 50 mM potassium chloride solution

Formulation IVa

-   1.0 g Fotecoat 1010 emulsion (FOTEC AG)-   0.05 g 0.1M ferrocene-   0.05 g 0.1M ferrocinium-   20 microL 50 mM potassium chloride solution

Formulation IVb

-   Oil:-   1.0 g 10% acrylated siloxane (Rhodia, Rhodosil R01194, 800 cSt)-   0.025 g α-hydroxycyclohexylphenylketone (Aldrich, 405612)-   0.025 g αα-dimethyl-α-phenylacetophenone (Fluka, 38781)-   Water:-   0.053 g polyvinylalcohol (Fluka, 18-88), derivatized with    2.75%(+/−0.25%) SBQ-   1.3 g DI water-   21 microL 50 mM potassium chloride solution

Formulation IVc

-   Oil:-   1.0 g 10% acrylated siloxane (Rhodia, Rhodosil R01194, 800 cSt)-   0.025 g α-hydroxycyclohexylphenylketone (Aldrich, 405612)-   0.025 g αα-dimethyl-α-phenylacetophenone (Fluka, 38781)-   Water:-   0.052 g polyvinylalcohol (PolyScience 49-88)-   1.3 g DI water-   10 microL 50% glutaraldehyde aqeuous solution-   0.052 g 0.1M potassium ferricyanide solution-   0.052 g 0.1M potassium ferrocyanide solution-   21 microL 50 mM potassium chloride solution

In initial screening experiments at room temperature in a flow cell, allformulation families except those with SBQ derivatized polyvinylalcoholgave acceptable reference electrode performance (wet-up less than 100seconds, minimal residual liquid junction potential). Formulations withSBQ derivatized polyvinylalcohol exhibited significant response tochloride, presumably being due to the ion exchange properties of the SBQcation which is part of the hydrophilic compartment's cross-linkingsystem. This data demonstrates that while the most straightforwardmethod of reducing salt diffusion coefficient is by cross-linking of thehydrophilic matrix, the cross-linking chemistry may impart deleteriousperformance characteristics.

Although cross-linking of the hydrophilic compartment is a possibleapproach, we have found that cross-linking the hydrophobic compartmenthas been a more generally successful approach. Cross-linked acrylatederivatized siloxanes generally gave good results, with formulationscontaining less than 10% acrylate derivitization generally beingsuperior. Highly acrylated siloxane formulations resulted in slower andmore variable wet-up characteristics, particularly at 37° C.

In the above formulations, potassium chloride was often added to thehydrophilic compartment of membranes also loaded with potassium ferroand ferricyanide redox salts. It appears that the addition of potassiumchloride does not significantly improve the performance of themembrane's salt bridge. We have found that the redox salts on their own,without additional other salts impart good salt bridge properties(either when using potassium ferrocyanide or potassium ferricyanidealone or in mixtures). This is presumably because the redox saltsthemselves are approximately equi-transferrent. We have found that theaddition of potassium chloride at high concentration actually candegrade the performance. The hydrophilic compartment containing highpotassium chloride content has variable drift characteristics and isshorter-lived because the additional salt causes the compartment toexcessively swell during wet-up and become too permeable to salttransport. In contrast, ferro and/or ferricyanide salts added at highconcentration may actually participate in cross-linking of thepolyvinylalcohol binder, thus reducing salt diffusion coefficient andimproving the use-life of the membrane. Even membranes prepared withoutany salt additions often give acceptable results. We infer from thisthat there are already some redox active contaminants in the membranepolymer systems (cross-linking agents, photoinitiators and the like)that can provide a low impedance interface with the gold electrode andpoise its potential, and that salts in the calibrator fluid whichpermeate into the membrane during wet up provide the salt-bridgeelectrolyte. Generally however the gold electrodes are better poisedwhen there is additional redox salt added to the membrane (less variableelectrode voltage during wet up), and salt-bridge potentials are lowerwhen the hydrophilic compartment has an approximately equi-transferentsalt composition.

Our preferred formulation was of type IIIa. The resulting heterogeneousmembrane comprised a hydrophobic compartment which was cross-linked 5%or 10% acrylated siloxane and the hydrophilic compartment comprisedpolyvinylalcohol binder. The hydrophilic compartment can contain one orboth of potassium ferrocyanide and potassium ferricyanide with noadditional salts. We have also prepared good membranes with very stablepotentials after wet up when the redox compound was the mixedferri-ferrocyanide, Prussian blue.

Oil and water components are gently mixed into a white 7-8 ml vial ofabout 15 mm diameter. The mixture is emulsified in the homogenizer atincreasing speeds, as described earlier. This formulation resulted inmembranes with a hydrophilic compartment (PVA) that is 5% by weight ofthe heterogeneous membrane. From gravimetric analysis we have estimatedthat after immersion of the membrane into an aqueous solution there is afew percent by weight water uptake into the membrane's hydrophiliccompartment. For equilibrium water uptake of less than 10% by weight ofthe dry hydrophilic compartment, the salt loading in the dry membranecorresponds with a concentration of about 1M or larger of potassiumferro and ferricyanide. Salts are thus loaded to be present at or inslight excess of their saturation solubility in the wetted-up membrane.

Membranes cast from the preferred emulsion formulation were in thethickness range of 0.005 to 0.01 cm. Membranes cast on a gold electrodeof an electrode module exhibited low noise and low resistance, wet up inunder 60 seconds, minimal residual liquid junction response tocompositional changes of the test solution and no redox interferences.When used as a reference electrode in combination with potentiometricindicator electrodes in a multi-sensor module in a diagnostic cardoperated at 37° C. we have obtained performance in conformance toclinically acceptable standards of precision and accuracy inmeasurements on whole blood.

Those skilled in the art of reference electrodes will recognize thatthere are many possible salt compositions that can be formulated to givea hydrophilic compartment containing approximately equi-transferrentelectrolyte yielding a salt bridge with a minimum residual liquidjunction potential. Such other formulations are possible so long as thehydrophilic compartment also contains redox species that react at theunderlying metal electrode which poise its potential, and so long as thesalt additions are compatible with a hydrophilic compartment havingsufficiently low salt diffusion coefficient that the salt bridge hasuseful lifetime. Those skilled in the art of gas permeable membraneswill recognize that there are many possible other materials for thehydrophobic compartment, so long as those materials can be formed into amembrane with an intimately admixed interpenetrating hydrophiliccompartment, and so long as those materials permit rapid water vaporpermeation.

Those skilled in the art will appreciate that the heterogeneous membraneof the invention can also be used with conventional reference electrodeelements. For example a salt bridge using the invented heterogeneousmembrane can be fabricated on a conventional silver—silver chlorideelectrode.

Prior Art Potentiometric Dissolved Carbon Dioxide Sensors

FIG. 2A shows a cross-section through a representative prior-art planarpotentiometric dissolved carbon dioxide sensor similar to one describedin the '184 patent. The device 80 which is part of a solid stateelectrode element in a disposable fluidic cartridge comprises a planarinsulating substrate 81, with conductor elements 82A and 82B on onesurface contacting two silver rod elements 83A and 83B with silverchloride over-layers 84A and 84B. One silver-silver chloride electrode83A/84A is the internal reference electrode the other 83B/84B becomesthe pH indicator electrode when coated with a thin film internalelectrolyte element 85 and a pH sensitive membrane 86. Two additionalhydrophilic matrix layers 87 and 88 containing chloride and bicarbonatesalts together constitute the integral internal electrolyte overlayingthe electrode pair. An outer gas permeable membrane 89 completes thesensor.

In use, the planar carbon dioxide sensor of the prior art is immersed inthe solution to be tested so that the solution contacts the outermembrane 89 of the sensor. In this device, typical of the classicalSeveringhaus type dissolved carbon dioxide sensor of the prior art, thecarbon dioxide is measured by the pH change within the hydrophilicelements 87 and 88. Carbon dioxide permeates through 89 and dissolvesinto layers 87/88 and is hydrolyzed to carbonic acid, which in turnionizes to bicarbonate ions and protons. As is known in the art, the pHchange in the internal electrolyte 87/88 measured by the voltage betweenthe contacts to the indicator electrode 82B and internal referenceelectrode 82A is proportional to the logarithm of the carbon dioxideconcentration change in the test solution when the bicarbonate andchloride concentrations in the internal electrolyte are constant.Non-volatile species are excluded from the internal electrolyteelectrode region by element 89.

Potentiometric Dissolved Carbon Dioxide Sensors with HeterogeneousMembrane

FIG. 2B shows a horizontal cross-section of a preferred embodiment ofthe present invention directed to potentiometric dissolved gaselectrodes, particularly to dissolved carbon dioxide electrodes. Theinvented device of FIG. 2B is remarkably simple when compared to thecomplex multi-layer device representative of the prior art. In theinvented device there is only one electrode as opposed to the electrodepair of the conventional Severinghaus type device. The electrode is ametal only (no metal salt as in the standard silver—silver chloridetechnology). The electrode metal is the same as the metal material ofthe electric contact. The various hydrophilic membranes and gaspermeable membranes used in prior-art devices are all now containedwithin either a single heterogeneous membrane coating of the metalelectrode(singly coated embodiment) or in a double coating comprising inaddition to the heterogeneous membrane a hydrophilic internal reservoirlayer interposed between the heterogeneous membrane and the metalelectrode (doubly coated embodiment). The electrode module 90 shown incross-section includes an insulating foil 91 laminated with a metal foilelement 92 and optional intermediate adhesive 93. A die-cut hole 94through the insulator foil 91 determines the location of the electrode.The membranes 95 include at least a heterogeneous membrane comprising anintimate admixture of a hydrophobic polymeric compartment that is watervapor and carbon dioxide permeable (but not permeable to electrolyte)and a hydrophilic, electrolyte permeable compartment.

In the singly coated embodiment, the heterogeneous membrane'shydrophilic compartment constitutes the internal reagent reservoir whichcontains at least a bicarbonate salt and a pH sensitive redox couplethat undergoes pH dependent reversible oxidation-reduction at the metalelectrode. In a specific preferred embodiment of the single membranedevice the electrode is gold, the heterogeneous membrane consists ofpolydimethylsiloxane hydrophobic polymer intimately admixed with ahydrophilic compartment that comprises a cross-linked polyvinylalcoholcontaining bicarbonate salt and quinhydrone. Other optional componentsare carbonic anhydrase, other electrolyte salts and surfactants.

In the doubly coated embodiment the internal reservoir layer interposedbetween the heterogeneous membrane and the electrode now contains atleast bicarbonate salt and a pH sensitive redox couple, and optionallyalso carbonic anhydrase. In a preferred embodiment of the doubly coatedelectrode the internal reservoir layer is polyvinyl alcohol containingbicarbonate salt, quinhydrone salt and optional carbonic anhydrase. Theheterogeneous membrane comprises a hydrophobic compartment with photocross-linked acrylated siloxane (preferably less than 5% acrylated) anda hydrophilic compartment with polyvinyl alcohol (preferably less than5% by volume of the heterogeneous membrane).

In use of the carbon dioxide sensor in accordance with the invention,electrical contact is made to the lower contact metal surface of themodule by an external measuring circuit, thus contacting the indicatorcarbon dioxide electrode and a salt bridge reference electrode (also onthe module but not shown in the above diagram). The upper surface of themodule is first immersed in calibrator solution so that the solution isin contact with the outer heterogeneous membrane 95 of the sensor then,after a time t, it is immersed in a test solution whose PCO₂ is to bedetermined. When immersed in the calibrator solution, the heterogeneousmembrane and the internal reagent reservoir wet up by water absorptionthrough the hydrophobic compartment of the heterogeneous membrane, thenby equilibrium partitioning from the hydrophobic compartment to thehydrophilic compartment and the internal reservoir. Electricalcontinuity between the indicator electrode and the external salt-bridgereference electrode is provided by electrical conduction through theheterogeneous membrane's hydrophilic compartment. Carbon dioxide in thecalibrator solution also permeates the membrane by diffusion through thehydrophobic compartment, then by equilibrium partitioning from thehydrophobic compartment into the hydrophilic compartment and theinternal reservoir. Carbon dioxide dissolves in the water within theaqueous pores of the hydrophilic compartment or the internal reservoirlayer containing bicarbonate salt and pH dependent redox couple, whereit hydrolyses forming hydrogen ions in accordance with the equilibriumrelation shown in the following equation

where K₁ and K₂ are the first and second dissociation constants ofcarbonic acid. A first pH established at the membrane's inner surfaceduring immersion in calibrator leads to a first measurable electrodevoltage, which voltage is related to the known PCO₂ in the calibratorsolution. At time t the calibrator solution is removed and a testsolution is brought in contact with the membrane. At this time a secondelectrode voltage corresponding to a second pH in turn related to theunknown PCO₂ in the test solution is measured. The measured milivoltresponse resulting from the pH change at the membrane's inner surface isrelated only to the PCO₂ concentration change between the calibrator andtest solutions so long as the bicarbonate concentration at themembrane's inner surface is approximately constant through the period oftime that the milivolt electrode responses are measured.

The hydrophobic gas permeable compartment of the heterogeneous membraneshould be present in sufficient quantity to achieve sufficient and rapid(typically less than 60 seconds) water uptake into the initiallysubstantially dry membrane during the calibration step, and to permitrapid equilibration of the heterogeneous membrane to the change incarbon dioxide concentration as the immersing solution is transitionedfrom calibrator to test solution.

During and after wet-up of the invented electrode there is continuousdepletion of the heterogeneous membrane of those reagents initiallyincorporated into its hydrophilic compartment or its internal reservoirlayer (bicarbonate salt and pH dependent redox electrolytes) byout-diffusion into the calibrator fluid. The concentration of thesereagents in the heterogeneous membrane decreases through this time. Theinitial quantities of reagents in the membrane, the membrane's thicknessand the reagents' diffusivity within the membrane's hydrophiliccompartment determine the rate of change of reagent concentrations andthe time to deplete the reagents to a critical threshold concentrationlevel and the time to introduce contaminants to a critical concentrationlevel, contaminants being buffers or redox contaminants that mightinterfere with the measurement. At the time t at which the test solutionis applied to the electrode the reagent concentrations within themembrane should be at or above the required threshold concentration, andcontaminants below a required threshold level at which the electrode'sPCO₂ response slope is known and reproducible. Notably, the bicarbonateconcentration should be in excess of the concentration of pH bufferingmoieties (but not larger than about 800 mM, at which concentration thereis also appreciable carbonate and the electrode's response slope isdepressed). In other words, the optimally performing device will exhibita reproducible response slope to a change in the dissolved carbondioxide concentration between the calibrator and the test solution up toa time t at which the bicarbonate concentration is in excess of buffercontaminants, and the pH dependent redox reagent is at a sufficientconcentration excess over redox contaminants to constitute the potentialdetermining electrode reaction.

The optimally performing device should also exhibit a speed of responseto the change in the carbon dioxide concentration going from calibratorto test solution (which is the sensor signal) that is fast compared tothe slower speed of response due to changes of other membrane reagentconcentrations (the potential determining pH dependent redox electrolyteor the pH determining bicarbonate salts) as they diffuse out from theheterogeneous membrane and fast compared to the slower response due tocontaminants (buffers or redox active species) diffusing into themembrane. Both the slow influx of contaminants and slow efflux ofmembrane reagents constitute an electrode drift during the time oftransition between calibrator and test solutions. So long as thesignal's time response is fast compared to these electrode driftresponses the signal can be accurately extracted from the drift. Toassure these conditions, it is preferred that the membrane's diffusioncoefficient of carbon dioxide be much larger than the diffusioncoefficient of the electrolyte salts initially loaded into the membrane.A heterogeneous membrane formulated with a low salt diffusioncoefficient also impedes the transport of redox contaminants, protons orbuffers from the test solution to the electrode surface where they mightcompete as the potential determining electrode reactants or where theymight alter the internal pH and interfere with the pH determined by thehydrolysis of dissolved carbon dioxide.

Dissolved Carbon Dioxide Electrode Examples:

To further understand the design rules for formulating the heterogeneousmembrane of the dissolved carbon dioxide sensor according to thisinvention we present a number of exemplar membrane formulations andtheir sensor performance

The preferred embodiments of the carbon dioxide electrodes in accordancewith the invention are fabricated with a heterogeneous membrane coatingstep on top of a metal electrode which has a first coating of aninternal reservoir layer. This reservoir layer comprises a hydrophilicmatrix with the reservoir salts, bicarbonate and pH dependent redox saltand also containing carbonic anhydrase. It is also feasible to makecarbon dioxide electrodes with only a single heterogeneous membranecoating the metal electrode. This requires the heterogeneous membrane'shydrophilic compartment to act as the internal salt reservoir containingbicarbonate and pH dependent redox reagent. In either case theheterogeneous membrane's gas permeable compartment permits water vaportransport to allow rapid wet-up of the internal reservoir, whether it beincorporated in a separate internal reservoir layer or as part of theheterogeneous membrane's hydrophilic compartment. The heterogeneousmembrane's gas permeable path also permits rapid transport of carbondioxide from the test solution to the internal reservoir where thecarbon dioxide dissolves and changes the internal reservoir's pH. Thehydrophilic compartment of the heterogeneous membrane permits transportof salts between the internal reservoir and the test solution toestablish a liquid junction and provide electrical continuity to enablea potentiometric measurement versus an external reference electrode.

The preferred carbon dioxide electrodes comprised an inner reservoirlayer formulated either with a chemically cross-linked polyvinylalcoholbinder, or one that is not chemically cross-linked, as shown in theexemplar formulations recited below

Cross-Linked Internal Reservoir:

-   0.07 g polyvinylalcohol (Fluka, 18-88), derivatized with    2.75%(+/−0.25%) SBQ 1.63 g DI water-   0.1 g 0.1M benzoquinone (Sigma) solution-   0.1 g 0.1M hydroquinone (Sigma) solution-   0.22 g 0.2M sodium bicarbonate (Sigma) solution-   Addition of sodium bicarbonate is performed with vortexing

Non Cross-Linked Internal Reservoir:

-   0.1 g polyvinylalcohol (PolyScience, 56-98)-   1.15 g DI water-   0.8 g 0.1M benzoquinone (Sigma) solution-   0.11 g 0.1M hydroquinone (Sigma) solution-   0.05 g 1M sodium bicarbonate (Sigma) solution-   9 microL of 4% by weight carbonic anhydrase (Sigma) solution

The benzoquinone to hydroquinone ratio need not be 1:1 as in theclassical quinhydrone redox couple. The amount of hydroquinone loadingis less critical than benzoquinone, indeed it can be completely absent.Generally higher concentrations of benzoquinone are preferred.Formulations were also made using other quinone based pH sensitive redoxmolecules of the known art such as thymoquinone in place ofbenzoquinone, giving similar results.

The heterogeneous membrane coating over the internal reservoir can beeither cross-linked in the hydrophilic compartment using SBQ derivitizedpolyvinylalcohol, or it can be formulated with a cross-linkedhydrophobic compartment as recited in the formulations below,cross-linking being photo initiated.

Polydimethylsiloxane/PVA-SBQ Heterogeneous Membrane Layer:

-   Oil:-   1.5 g polydimethylsiloxane (Aldrich, 378402, 10,000 cSt)-   0.5 g hexamethyldisiloxane (Aldrich, 205389)-   Water:-   0.06 g polyvinylalcohol (Fluka, 18-88), derivatized with    2.75%(+/−0.25%) SBQ-   1.22 g DI water-   0.2 g 0.2M potassium chloride solution-   0.21 g 0.2M sodium bicarbonate solution    Acrylated siloxane/polyvinylalcohol Heterogeneous Membrane Layer:-   Oil:-   2.0 g 5% acrylated siloxane (Gelest, USC-052, 150-200 cSt)-   0.05 g α-hydroxycyclohexylphenylketone (Aldrich, 405612)-   0.05 g αα-dimethyl-α-phenylacetophenone (Photo initiator, Fluka,    38781)-   Water:-   0.075 g polyvinylalcohol (Poly Science 49-88)-   1.5 g DI water

The most preferred formulation for the heterogeneous membrane has usedphoto cross-linked acrylated siloxane formulations, the degree ofacrylation being less than 5%.

We have used the quinhydrone couple (hydroquinone plus benzoquinone) asthe pH dependent redox salt, but other pH dependent redox salts areknown in the art and could also be used. (see for examples. J. Slatteryet al. Coordination Chemistry Reviews 174, (1998) 391-416).

Experimental wet-up transients agree well with our computations(discussed below and shown in FIG. 3A). There is an initial wet upperiod (typically about 60 seconds or less) during which the electrodevoltage increases rapidly as the dry bicarbonate in the internalreservoir acquires water and its pH decreases. A plateau is thenachieved at which time the voltage increases more slowly as bicarbonateslowly diffuses out of the reservoir and its pH decreases slowly. Wehave targeted a dry bicarbonate salt loading which achieves an internalreservoir concentration in the range of 100 mM to 200 mM after membranewet up. We can confirm that the target concentration has been achievedin the experimental membrane electrodes by observing their measuredelectrode potential after wet up, and knowing the pH dependence of thequinhydrone electrode we can compute the pH of the internal reservoir,and thus the bicarbonate concentration.

In the preferred embodiment of a singly coated carbon dioxide electrodeusing only a single heterogeneous membrane coating on the electrodethere is no additional internal reservoir layer, and the bicarbonatesalt, pH dependent redox salts and carbonic anhydrase are loaded intothe hydrophilic compartment of the heterogeneous membrane which nowconstitutes the internal reservoir.

To better understand the desirable transport properties of the membraneof the electrode in accordance with the invention, we have generateddesign parameters based on simulations of the device's performance.Using a numerical finite element analysis of diffusion we computed thetime and position transient species concentrations within theelectrode's heterogeneous membrane. We computed the transientconcentration of water, carbon dioxide, bicarbonate and theconcentration of contaminating buffers at the membrane's inner surfacecontacting metal electrode versus time for different membrane saltdiffusion coefficients, initial bicarbonate salt loading in the membraneand the membrane thickness. In these computations we simulated typicalmembrane formulations and dimensions that were investigatedexperimentally, comprising a polydimethylsiloxane hydrophobiccompartment and a polyvinylalcohol hydrophilic compartment containingsalts. We simulated membrane thicknesses in the range 80+/−20micrometers. We modeled a heterogeneous membrane comprising 95%-98% byvolume of a polydimethylsiloxane hydrophobic compartment with atortuosity of 2 giving a membrane gas diffusion coefficient of 5×10⁻⁶for both water vapor and carbon dioxide, with solubility of 1×10⁻³ and6×10⁻⁵ moles/cm³/atm. for water vapor and carbon dioxide respectively.We assumed a hydrophilic compartment whose equilibrium water uptake wasin the range 0.01 to 0.2 (total liquid water volume per membrane volumeafter wet-up being in the range 0.01×2% to 0.2×5%=0.02% to 1%). Weassumed that carbon dioxide dissolved in the pore water of thehydrophilic compartment with a solubility of 2.3×10⁻⁵ moles cm⁻³ atm.⁻¹.

We considered an initially dry heterogeneous membrane electrode immersedin an aqueous solution. We computed the transient concentrations ofwater as the membrane wets up, of carbon dioxide, and of various salts:bicarbonate and pH dependent redox salts initially loaded into themembrane as they diffused out of the membrane into calibrator solution,and the concentration of contaminants (buffers, acids, bases and redoxactive species) as they diffused in. Our simulation computed thesetransient concentrations during the time period of initial wet-up in thecalibrator liquid and the time when the calibrator is removed and a testsolution is introduced to the electrode.

From this analysis we obtained species concentrations in the hydrophiliccompartment at the inner membrane surface versus time. From thesecomputed concentrations we could determine the electrode's carbondioxide response slope. At the membrane's inner surface at time t thedissolved carbon dioxide at concentration C_(dCO2) is in equilibriumwith the bicarbonate and carbonate salts at concentrations of C_(HCO3−)and C_(CO3−). The proton concentration C_(H+) (and pH given bypH=−LOG₁₀C_(H+)) of the hydrophilic compartment of the heterogeneousmembrane at the inner boundary changes with dissolved carbon dioxideconcentration and bicarbonate salt and buffer salt concentrations, whichchange can be computed from the following equilibrium equations:

the equation of mass balance for carbon containing species

C_(NaHCO3)═C_(HCO3−)+C_(dCO2)+C_(CO3−)  Equation 4

the equation of mass balance for buffer species

C_(HB)+C_(NaB)═C_(TB)   Equation 5

the charge balance equation

C_(NaHCO3)+C_(NaB)═C_(B−)+C_(HCO3−)+2C_(CO3−)  Equation 6

the 1st dissociation of carbonic acid

C_(HCO3−)═K₁ (C_(dCO2)/C_(H+))   Equation 7

the 2nd dissociation of carbonic acid

C_(CO3−)═K₂ (C_(HCO3−)/C_(H+))=K₁K₂ (C_(dCO2)/C_(H+) ²)   Equation 8

the buffer equilibrium equation

C_(B−)═C_(TB)/(1+(C_(H+)/K_(B)))   Equation 9

The electrode potential is the sum of the potential difference betweenthe electrode and the electrolyte in the hydrophilic compartment at theinner boundary due to the potential determining pH dependent redoxreaction at the electrode surface plus the liquid junction potentialbetween the membrane and the test solution. The potential at theelectrode surface is determined by the pH in accordance with theequilibrium equation of the pH dependent redox couple. Using quinhydroneas example

2H⁺+Q+2e

H₂Q   Equation 10

where the oxidant is benzoquinone (Q) and the reductant is hydroquinone(H₂Q), the electrode potential is given by

$\begin{matrix}\begin{matrix}{V = {V_{QH} + {\frac{kT}{2q}{Ln}\frac{C_{Q}C_{H +}^{2}}{C_{H_{2}Q}}}}} \\{= {V_{QH} + {\frac{kT}{2q}{Ln}\frac{C_{Q}}{C_{H_{2}Q}}} + {\frac{kT}{q}{{Ln}C}_{H +}}}} \\{= {{Const} - {0.06\mspace{14mu} {pH}}}}\end{matrix} & {{Equation}\mspace{14mu} 11}\end{matrix}$

where C_(Q) and C_(H2Q) are the concentrations of the benzoquinone andhydroquinone.

We have computed the hydrogen ion concentration and thence the electrodemilivolt response from the above quasi-equilibrium equations fordifferent concentrations of carbon dioxide, bicarbonate and buffer saltsin the membrane at the electrode surface at a time t after thecommencement of the measurement, these concentrations being determinedfrom the finite element analysis of diffusion. FIG. 3 shows a series ofexemplar simulated voltage transients of electrodes of the invention. Inthis simulation we computed the response of three membranes, each loadedinitially to a concentration of 400 mM sodium bicarbonate (calculated asthe number of moles of dry bicarbonate salt initially loaded into themembrane divided by the volume of pore water at equilibrium wet-up). Inthe simulation, the membrane was initially immersed in a calibratorsolution containing pCO₂ at 30 mm Hg, 30 mM bicarbonate and 50 mM ofbuffer comprising equal concentration of the buffer acid and thebuffer's sodium salt and a pK of 7.4. At time t=150 seconds the membranewas immersed in a test solution containing PCO₂ at 10 mm Hg, bicarbonateat 30 mM and total buffer at 15 mM. We computed the voltage transientsfor three different salt diffusion coefficients: curve A at 1×10⁻⁷,curve B at 3×10⁻⁷ and curve C at 1×10⁻⁶ cm²/sec. The transients show aninitial period of about 60 seconds of wet-up. At 60 to 150 seconds thereis a monotonic voltage drift associated with slow bicarbonate efflux andbuffer influx. The drift rate is larger for larger salt diffusioncoefficients. At 150 seconds, when there is a switch from the calibratorto a test solution with a different PCO₂, the electrode responds to thePCO₂ change. The magnitude of the response is determined by the saltcomposition of the membrane's hydrophilic compartment at the innersurface at that point in time. As shown in the simulation, the membranewith a large salt diffusion coefficient (curve C) has been substantiallydepleted of bicarbonate and substantially contaminated with buffer sothat the carbon dioxide response slope is diminished. We have repeatedthis computation for many membrane formulations with differentbicarbonate loading and salt diffusion coefficients to furtherillustrate how the carbon dioxide response slope is affected by theseparameters.

The graph of FIG. 4 shows the carbon dioxide response slope (milivoltsoutput per decade change of PCO₂ , for a transition form 30 mm Hg in thecalibrator to 10 mm Hg in the test solution) versus bicarbonate andbuffer concentration at the membrane's inner surface at time t when theswitch from calibrator to test solution is made. This graph teachesthat, as the bicarbonate content of the membrane is increased, the pH atthe membrane's inner surface becomes more basic, the concentration ofcarbonate increases and the response slope is reduced. Thus there is anupper threshold for the preferred bicarbonate concentration that givesthe best response slope. Using a cut-off of 48 mV/decade (0.8 of Nernstslope) as the minimally acceptable slope (corresponding to an acceptablerange of 52+/−2 mV/decade we can specify the required bicarbonateconcentration at time t. This preferred concentration of bicarbonate ofthe fully wet-up membrane at the inner boundary should be less thanabout 800 mM at the time t of measurement of the test solution. Thisgraph also teaches that at low bicarbonate concentration in themembrane, the response slope is diminished as the concentration ofcontaminating buffer is increased. The amount of buffer is determined bythe sum of that which has permeated into the membrane from thecalibrator solution and any buffer contaminant incorporated initiallyinto the membrane. Typically, a hydrophilic membrane binder such aspolyvinylalcohol will contain proton binding sites which constituteinternal buffers that are part of the membrane's hydrophiliccompartment. Compositions of membranes with large internal bufferconcentrations should be avoided to obtain good electrode response slopeover a wide range of bicarbonate loading. The preferred minimumbicarbonate concentration for good electrode response is about 50 mM inthe presence of buffer salts at a concentration of up to about 50 mM. Abicarbonate concentration of about 100 mM at the electrode surface atthe time of measurement gives a CO₂ response slope in the range 52 to 56mV per decade. Membranes with larger internal buffer concentrations canbe tolerated, but the bicarbonate salt loading must be increased so thatthe bicarbonate concentration is in excess of buffers at the time ofmeasurement.

TABLE 3 D cm²/sec 1 × 10⁻⁷ 2 × 10⁻⁷ 5 × 10⁻⁷ thickness cm 0.01 0.0080.006 0.01 0.008 0.006 0.01 0.008 0.006 [HCO₃ ⁻] 100 secs <0.8 <0.8 0.850.80 0.84 0.91 0.88 0.93 0.83 800 mM 200 secs 0.82 0.85 0.91 0.87 0.920.91 0.93 <0.8 <0.8 300 secs 0.85 0.89 0.94 0.92 0.93 <0.8 <0.8 <0.8<0.8 100 secs 0.85 0.88 0.90 0.87 0.91 0.94 0.93 0.94 <0.8 400 mM 200secs 0.89 0.91 0.94 0.92 0.94 <0.8 0.89 <0.8 <0.8 300 secs 0.91 0.930.92 0.94 0.90 <0.8 <0.8 <0.8 <0.8 100 secs 0.91 0.93 0.95 0.93 0.950.94 0.94 0.90 <0.8 200 mM 200 secs 0.93 0.94 0.93 0.95 0.93 <0.8 0.83<0.8 <0.8 300 secs 0.94 0.94 0.88 0.93 0.87 <0.8 <0.8 <0.8 <0.8In conclusion the preferred bicarbonate loading of the membrane isbetween 50 mM and 800 mM at the time of measurement.

We have computed the transient response of heterogeneous membraneelectrodes when there is a transition from calibrator to test fluid at atime t after the initial immersion of the electrode in calibrator.Typical computations are shown in FIG. 5. In this simulation, aninitially dry heterogeneous membrane electrode is initially loaded withsodium bicarbonate and quinhydrone. The electrode is first immersed in acalibrator solution whose composition is PCO₂=30 mm Hg, concentration ofbicarbonate at 30 mM and buffer (pK=7.5) concentration of 50 mM, thenimmersed in a test solution with PCO₂=100 mm Hg, at differentbicarbonate concentrations spanning the clinical range from 10 to 60 mM,and 15 mM buffer. The transient response when switching betweencalibrator and test solutions at t=150 seconds at constant bicarbonateconcentration is curve A showing a response time of about 30 seconds toPCO₂ superimposed on a monotonically drifting background. The backgrounddrift is associated with the continuous slow efflux of the bicarbonateinitially loaded into the membrane from the fully wet-up membrane. Thesignal (S) is the milivolt response to the change in PCO₂ betweencalibrator and test solutions. The same device when exposed to a testsolution with high bicarbonate concentration responds according to curveB, and low bicarbonate concentration curve C. The difference betweenthese voltage transients at the point in time that the electrode hasfully responded to the PCO₂ change is the bicarbonate interference I.The different voltage transients result because during the time afterthe fluid switch when carbon dioxide diffuses into the membrane toestablish a new equilibrium pH at the inner membrane surface thebicarbonate in the test solution also diffuses into or out of themembrane and affects the membrane's internal pH. The degree to whichthere is bicarbonate interference is determined by the relative rate ofdiffusion of carbon dioxide gas and bicarbonate salt. This in turndepends on the relative diffusion rates of gas and salt and the totalinitial bicarbonate loading.

To further illustrate this we have computed the bicarbonate interferencefor a range of membranes with different initial salt loading, saltdiffusion coefficient. We have computed the bicarbonate interference (I)in units of % change of PCO₂ per 10 mM change in bicarbonateconcentration. The membrane thickness was 0.008 cm. These simulated dataare shown in the table below.

TABLE 4 Secs 1 × 10⁻⁷ 2.5 × 10⁻⁷ 5 × 10⁻⁷ 100 0.01 0.5 4.7 800 mM 2000.02 1.4 10.3 300 0.03 2.5 11.1 100 0.03 1.0 7.5 200 0.04 2.1 10.9 400mM 300 0.06 2.9 11.2 100 0.06 1.7 10.1 200 0.08 2.8 11.2 200 mM 300 0.113.3 11.2The conclusions from the above simulation data are:

-   -   For a membrane with a carbon dioxide gas diffusion coefficient        of 5×10⁻⁶ cm²/sec the marginally acceptable salt diffusion        coefficient is 5×10⁻⁷ cm²/sec, and then only when the initial        bicarbonate loading in the membrane is high (<800 mM) and the        measurement time is short (t<100 secs). This corresponds with a        minimum diffusion constant ratio, D_(gas)/D_(salt) of about 10.        A faster responding carbon dioxide response is tolerant to a        faster bicarbonate response, but the minimally acceptable ratio        of diffusion coefficient remains the same.    -   Preferred membranes have a diffusion coefficient ratio of 20 at        which ratio there is lower bicarbonate interference, and still        more preferred is 50 or larger, at which ratio there is no        resolvable bicarbonate interference.

Prior Art Polarographic Oxygen Sensors

FIG. 6A illustrates a representative planar polarographic Clarke typeoxygen sensor of the prior art. The device 100 shown in cross-sectionconsists of a planar insulating substrate 101 supporting a metal layer102 formed into two conducting elements 102A and 102B, and an insulatinglayer 103 overlaying them. Openings 104A and 104B through the insulatinglayer define the position of two electrodes, an indicator electrode andan internal reference electrode. Elsewhere on conductors 102A and 102B acontact is made to an external measuring circuit. Conductor element 102Ais coated by films of silver and silver chloride formed into elements105 and 107 constituting the internal silver/silver chloridereference-counter electrode. Conductor element 102B is coated by a filmof gold formed into an electrode element 106 which is the indicatorelectrode. A film of a hydrophilic electrolyte medium 108 covers bothelectrodes. Electrolyte film 108 provides electrical continuity betweenelectrodes at 104A and 104B. A film of a gas permeable, electrolyteimpermeable material is formed into a cover element 109 that coatselectrolyte film 108.

In use, the illustrative planar device of the prior art is immersed inthe solution to be tested so that the solution contacts the outermembrane 109 of the sensor. Oxygen dissolved in the test solution istransported through gas permeable element 109 into the internalelectrolyte reservoir 108 to the polarographic indicator electrode at104B. Non-volatile electro-active species are excluded from theelectrode region by layer 109. In this device, typical of the classicalpolarographic dissolved oxygen electrode of the prior art, the oxygenconcentration is analyzed by measuring the oxygen reduction at the goldelectrode. Typically, a cathodic voltage of several hundred milivolts isapplied to the gold electrode versus the internal reference electrode.Electrical continuity between internal reference electrode and thecathode is through the internal reservoir layer 108 which iselectrically isolated from the test solution by layer 109. As is knownin the art, the current flowing between the two electrodes isproportional to the diffusion current of oxygen to the reducingelectrode, which in turn is proportional to the oxygen concentration inthe test solution.

Polarographic Oxygen Sensors with Heterogeneous Membrane

The invented device of FIG. 6B is remarkably simple when compared to thecomplex multi-layer device representative of the prior art. In theinvented device there is only one electrode. The electrode is a metalonly (no metal salt as in the standard silver—silver chloridetechnology). The metal is the same as the metal contact material. Thevarious hydrophilic membranes and gas permeable membranes used inprior-art devices are all now contained within a single heterogeneousmembrane. The electrode module 110 shown in cross-section includes aninsulating foil 111 laminated with a conducting metal foil element 112and optional intermediate adhesive 113. A die-cut hole 114 through theinsulator foil 111 determines the location of the electrode. Theheterogeneous membrane 115 consists of a hydrophobic polymericcompartment that is water vapor and oxygen permeable (but not permeableto electrolyte) and a hydrophilic, electrolyte permeable compartment. Ina preferred embodiment of this device the electrode is gold, theheterogeneous membrane consists of a cross-linked hydroxyl derivatizedepoxy hydrophobic polymer admixed with a hydrophilic compartment thatcomprises cross-linked polyvinylalcohol. Additional optional componentsof the hydrophilic compartment of the membrane are surfactants, buffersand electrolyte salts.

In use of the invented polarographic oxygen sensor, electrical contactto an external measuring circuit is made to the lower contact metalsurface of the module. The upper surface is immersed in calibratorsolution so that the solution is in contact with the outer heterogeneousmembrane 115 of the sensor. The heterogeneous membrane wets up by waterabsorption through the hydrophobic compartment of the membrane, then byequilibrium partitioning from the hydrophobic compartment to thehydrophilic compartment. Oxygen in the calibrator solution alsopermeates the membrane by diffusion through the hydrophobic compartment,then by equilibrium partitioning from the hydrophobic compartment intothe hydrophilic compartment including the hydrophilic compartment at thesurface of the metal electrode, which constitutes the sensor's internalreservoir. A cathodic voltage of several hundred millivolts is appliedto the electrode versus an external reference-counter electrode (notshown). Electrical continuity between the sensor's electrode at 112 andthe solution containing the external reference/counter electrode is byelectrical conduction through the hydrophilic compartment of theheterogeneous membrane 115. Electrolyte transport through thehydrophilic compartment of the heterogeneous membrane 115 also permitsout-diffusion of salts and other non-volatile reagents from the surfaceof electrode element 112 and in-diffusion of contaminants andinterferents from the test solution, but their rate of diffusion beingsufficiently slow that they do not reach a concentration sufficient tocause erroneous oxygen measurement during the time of the use of thedevice. This behavior is in marked contrast to prior-art devices. Theoxygen dissolved in the hydrophilic compartment at the membrane's innersurface is reduced at the cathodic electrode. The reduction current isproportional to the oxygen concentration at the inner surface which isalso proportional to the known concentration in the calibrator solution.At a time t the calibrator solution is removed and a test solution isbrought into contact with the sensor's membrane. The oxygenconcentration in the hydrophilic compartment at the membrane's innersurface changes to a new value proportional to the concentration ofoxygen in the test solution, the cathodic electrode current now beingproportional to the concentration of oxygen in the test solution.

In a preferred formulation of the heterogeneous membrane in accordancewith the invention, the hydrophilic compartment of the heterogeneousmembrane is confined to a small fraction of the total membrane volume,typically about 5% by volume or less, and the permeability of thehydrophilic compartment to redox active chemicals in the test solutionis sufficiently small so that the electrode current due to interferingredox reactions is small compared to the signal current due to reductionof the dissolved oxygen being analyzed. The lower limit for the volumefraction of the hydrophilic compartment of the heterogeneous membrane isdetermined by the requirement for electrical continuity across themembrane element. Under normal measurement circumstances theheterogeneous membrane's bulk resistance should be less than about 10⁸ohm to assure electrical continuity, not to incur a significant voltagedrop through the membrane's thickness, and to have immunity from noise.

The oxygen permeability of a preferred heterogeneous membranecomposition should be sufficiently low so that oxygen conductancethrough the membrane is lower than through the fluid above the membrane.With this condition there is minimal concentration polarization in thefluid and the electrode's oxygen response is not dependent on thefluids's flow rate or its hydrodynamic mixing. Also, a heterogeneousmembrane whose hydrophobic compartment comprises a material with highoxygen permeability will likely also have large oxygen solubility. Suchmembranes are slower to respond and are therefore not favored. Toestimate the upper limit of the desirable oxygen permeability of themembrane we first calculate oxygen conductance through the aqueous fluidabove the membrane. For a macro-electrode, this is given approximatelyby the planar diffusional flux per unit area per unit pressure. Theconductance is given by C=P/x, where P is the permeability of oxygen inthe fluid through a diffusion layer of thickness x, x being in the range0.005 cm (flowing fluid)≦x≦0.05 cm (stagnant fluid). The oxygenpermeability P through an aqueous fluid is the diffusion coefficient(2×10⁻⁵ cm² s⁻¹) times the solubility (1.5×10⁻⁶ mole cm⁻³ atm⁻¹) whichis P=3×10⁻¹¹ mole cm⁻¹ s⁻¹ atm⁻¹. This gives a conductance in the range6×10⁻¹⁰≦C≦6×10⁻⁹ mole cm⁻² s⁻¹ atm⁻¹. To avoid concentrationpolarization of the aqueous fluid above the membrane electrode, theconductance through the membrane, C_(m) , should be much smaller (say nomore than 20%) of the conductance through the aqueous fluid. This setsan upper limit on the membrane's conductance and thence also its oxygenpermeability P_(m) for a given membrane thickness d, given byC_(m)=P_(m)/d≦0.2×6×10⁻¹⁰=1.2×10⁻¹⁰ mole cm⁻² s⁻¹ atm⁻¹. For a membranewhose thickness is 5×10⁻³ cm, which is typical, the preferred maximumoxygen permeability is then about 6×10⁻¹³ mole cm⁻¹ s⁻¹ atm⁻¹. Thisresult teaches that heterogeneous membranes with hydrophobiccompartments comprising less oxygen permeable materials are moresuitable than those using siloxanes whose permeability exceeds thedesired upper limit (see Table I). Our formulation data described belowconfirm this finding. A heterogeneous membrane with a hydrophobiccompartment having high oxygen permeability can still be useful, butonly when the membrane's oxygen permeability can be reduced by a highlycross-linked hydrophilic compartment, so that the oxygen conductancethrough the highly cross-linked hydrophilic compartment at the electrodesurface becomes the rate determining transport step. In the alternativea highly cross-linked additional internal reservoir layer can beinterposed between the electrode and the heterogeneous membrane.However, membranes with too high permeability of their hydrophobiccompartment, having also high oxygen solubility, are still not preferredbecause they are slower to respond. In a preferred membrane whose oxygenconductance is C_(m)≦1.2×10⁻¹⁰ mole cm⁻² s⁻¹ atm⁻¹ immersed in anair-saturated calibrator fluid at 0.2 atmospheres oxygen, the oxygenflux to the electrode is 2.4×10⁻¹¹ mole cm⁻² s⁻¹ which corresponds withan electrode current density of about 1×10⁻⁵ amps cm⁻² (assuming 4electron cathodic reduction of oxygen).

Examples of Membranes for Oxygen Electrodes

To better understand the design rules for construction of polarographicoxygen electrodes according to this invention we present a number ofexemplar heterogeneous membrane formulations and their sensorperformance. Table 5 includes membrane formulations in which thehydrophobic compartment comprises a polymer system derived from a numberof different families. These include siloxanes, acrylate derivatizedsiloxanes, hydroxyl derivatized epoxies, polyvinylacetate and urethanes.Examples of membranes are given comprising of cross-linked hydrophobicpolymers, cross-linked hydrophilic polymers and both hydrophobic andhydrophilic polymers being cross-linked.

TABLE 5 Oxygen Hydrophobic membrane compartment Hydrophilic compartmentformulation # Non-crosslinked Non-crosslinked I — — Non-crosslinkedCrosslinked II polydimethylsiloxane SBQ derivatized a polyvinylalcoholpolydimethylsiloxane polyvinylalcohol with b ammonium dichromateCrosslinked Non-crosslinked III acrylated siloxane Polyvinyl alcohol aCrosslinked Crosslinked IV acrylated polyvinylalcohol/acrylate asiloxane crosslinked acrylated polyvinylalcohol/diazo b epoxy polyolcrosslinked polyvinylacetate polyvinylalcohol c

Formulation IIb

-   Oil:-   1.32 g polydimethylsiloxane (Sigma-Aldrich, 1,000 cSt)-   Water:-   0.71 g polyvinylalcohol solution (Fluka, 18-88- dissolved in DI    water to 21% solids)-   116 microL 1M ammonium dichromate solution-   37.5 microL 2M potassium chloride solution-   1.6 mL DI water    -   1. Dilute the polyvinylalcohol solution with DI water and salt        solutions.    -   2. Emulsify oil and water at 24,000 rpm for about 1 minute.    -   3. Print membranes, allow to dry at room temperature for about        15 minutes, then expose for 30seconds to low-power UV

Formulation IIIa

-   -   Oil:    -   1.48 g Zipcone-UA (100%-acrylated siloxane, Gelest)    -   Water:    -   1.71 mL DI water    -   0.105 g PEG(1000) diacrylate (1000 molecular weight polyethylene        glycol terminated at both ends with acrylate,        Polysciences—diluted to 48% in DIW and with 2.5%        2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone dissolved        in it (photoinitiator, Sigma-Aldrich))    -   0.923 g polyvinylalcohol (18-88, Fluka—dissolved in DI water to        19% solids) 43 microL 2M potassium chloride solution        -   1. Dilute the pre-dissolved PVA and PEG(1000) diacrylate            with the DI water, add potassium chloride solution, vortex.        -   2. Add the siloxane oil and emulsify at 6,000 to 8,000 rpm            for about 2 minutes, then at 24,000 rpm for about 1 minute.        -   3. Print membranes, allow to dry at room temperature for 15            minutes, then expose to UV (5 exposures of 2 seconds each).

Formulation IVa

-   -   Oil:    -   1.475 g Zipcone-UA (100%-acrylated siloxane, Gelest)    -   Water:    -   2.24 mL DI water    -   0.07 g PEG(1000) diacrylate (1000 molecular weight polyethylene        glycol terminated at both ends with acrylate,        Polysciences—diluted to 48% in DI water and with 2.5%        2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone dissolved        in it (photoinitiator, Sigma-Aldrich))    -   0.8 g polyvinylalcohol (18-88, Fluka—dissolved in DIW to 19%        solids)    -   0.15 g Diazo-LZ (polyvinylalcohol crosslinker, Esprix—dissolved        to 10% in DI water) 38 microL 2M potassium chloride solution        -   1. Dilute the pre-dissolved polyvinylalcohol and PEG(1000)            diacrylate with the DI water, add potassium chloride            solution, vortex.        -   2. Add the siloxane oil and homogenize at low speed for            about 2 minutes, then at top speed for about 1 minute.        -   3. Print membranes, allow to dry at room temperature for 15            minutes, then expose to UV (5 exposures of 2 seconds each).

Formulation IVb

-   Oil:-   1.625 g Ebecryl 6040 (Acrylated-Epoxy-Polyol, UCB)-   0.195 g Irgacure-500 (Photoinitiator, Ciba)-   0.048 g Irgacure-369 (Photoinitiator, Ciba)-   0.0075 g Zonyl FSN (Surfactant, DuPont)-   Water:-   1.888 g DI water-   1.079 g polyvinylalcohol (18-88, Fluka:dissolved in DI water to 14%    solids)-   0.15 g diacetone acrylamide (Reactive monomer, DSM Fine Chemicals)-   0.0075 g Dapro DF-900 (Defoamer, Elementis Specialties)-   0.012 g Diazo-DDAM-12 (polyvinylalcohol cross-linker, Materiali    Sensibili—dissolved to 3.1% in DI water)    -   1. Dilute the pre-dissolved polyvinylalcohol with the DI water,        then dissolve into it the diacetone acrylamide.    -   2. Add the rest of the ingredients and emulsify at 6,000 to        8,000 rpm for about 2 minutes, then at 24,000 rpm for about 1        minute.    -   3. Filter through 12 micrometer syringe filter.    -   4. Add Diazo cross-linker to filtered emulsion and mix.    -   5. Let sit for 1 hour to degas, then print membranes.    -   6. Let air-cure for 15 minutes then expose to UV for 4 seconds.

The epoxy polyols: Ebercyl 6040 and 608 also gave similar results. OtherIVb type formulations that we tested included acrylated urethanescopolymerized with polyols, giving similar results to the epoxies.Formulations based on blends of the acrylated epoxy-polyols withacrylated urethane-polyols also gave similar results.

Formulation IVc

-   -   1.575 g Vinac 285 (Polyvinylacetate emulsion, Air Products) 0.82        mL DI water    -   0.56 g polyvinylalcohol (18-88, Fluka—dissolved in DIW to 19%        solids)    -   0.332 g trimethylolpentane triacrylate (Sigma-Aldrich, with 1%        benzoin ethyl ether (photoinitiator, Sigma-Aldrich) dissolved in        it)    -   0.04 g dibutyl fumarate (plasticizer, Scientific Polymer        Products)    -   30 microL 2M potassium chloride solution        -   1. Vortex until homogeneous.        -   2. Print membranes, allow to dry at room temperature for            about 15 minutes, then expose to UV for 4seconds.

Electrodes were fabricated by micro-dispensing oil in water emulsionmembrane cocktails over the electrode orifice of an electrode module.For a typical device, the electrode orifice was a 0.08 cm diameter holein an epoxy foil overlaying a gold foil electrode, having an electrodearea of 5×10⁻³ cm². Approximately 1 mm diameter membranes were cast witha dry thickness in the range 2 to 5×10⁻³ cm. For an electrode of thisgeometry and a preferred current density of less than 1×10⁻⁵ amps cm⁻²in air-saturated calibrator the preferred maximum calibrator current ofthe electrodes is 5×10⁻⁸ amps.

Formulations in the IVb family were our preferred formulations. Allpreferred formulations meet the desired electrode performance criteriafor use in dissolved oxygen measurements in clinical applications. Whenused as an oxygen electrode in a multi-sensor module in a diagnosticcard operated at 37° C. we have obtained performance in conformance toclinically acceptable standards of precision and accuracy inmeasurements on whole blood.

Electrodes with preferred membrane formulations wet-up within 100seconds when they are 3×10⁻⁵ cm thickness or less. They have a currentdensity less than the desirable upper limit of 1×10⁻⁵ amps cm⁻² whenthey are thicker than 1.5×10⁻³ cm. Response time (100% response) tooxygen is 30 seconds or less when the membrane is less than 3×10⁻⁵ cmthickness. Therefore the preferred thickness range for the preferredmembrane formulations is between about 1.5×10⁻³ to 3×10⁻³ cm.

Those skilled in the art will recognize that many other biosensorelectrodes such as enzyme electrodes can be made with very simplemembrane construction when using the inventive principles.

1. A heterogeneous membrane for an electrochemical sensing electrode fordirect exposure to a sample, the membrane comprising a number of firsthydrophobic, gas permeable compartments; and a number of secondhydrophilic electrolyte salt permeable compartments, wherein the firstand second compartments are intimately admixed to form interpenetratingnetworks of hydrophobic and hydrophilic compartments.
 2. An electrodefor use in an electrochemical sensing device for the analysis of anaqueous sample, comprising an electric conductor; an insulating layer onthe conductor, the insulating layer having a through-going aperturedefining an electrode region; and a heterogeneous membrane as defined inclaim 1 for direct contact with the sample, the membrane in physicalcontact with the insulating layer in the electrode region and inelectrical contact with the conductor.
 3. The heterogeneous membrane ofclaim 1, wherein the hydrophobic compartments are in excess by volumeover the hydrophilic compartments so that a gas diffusion coefficient ofa gas species through the hydrophobic compartments is larger than a saltdiffusion coefficient of species dissolved in water.
 4. Theheterogeneous membrane of claim 3, wherein the gas diffusion coefficientis at least 10 times larger than the salt diffusion coefficient.
 5. Theheterogeneous membrane of claim 3, wherein the gas diffusion coefficientis at least 50 times larger than the salt diffusion coefficient.
 6. Theheterogeneous membrane of claim 1, wherein the heterogeneous membranehas a gas diffusion coefficient and a salt diffusion coefficient andwherein the gas diffusion coefficient is greater than 1×10⁻⁶ cm² s⁻¹ andthe salt diffusion coefficient is less than 1×10⁻⁷ cm² s⁻¹.
 7. Theheterogeneous membrane of claim 1, wherein the hydrophilic compartmentsconstitute less than 5% by volume of the total volume of theheterogeneous membrane.
 8. The heterogeneous membrane of claim 1,wherein the hydrophilic compartments constitute an internal reagentreservoir containing at least a bicarbonate salt and a pH sensitiveredox couple.
 9. The heterogenous membrane of claim 1, wherein thehydrophobic compartments include a polydimethylsiloxane hydrophobicpolymer and the hydrophilic compartment includes a cross-linkedpolyvinylalcohol containing bicarbonate salt and quinhydrone.
 10. Amethod of manufacturing a heterogeneous membrane as defined in claim 1in the form of an oil-in-water emulsion, comprising the steps ofdissolving components of the hydrophilic compartment in an aqueoussolution; premixing components of an oil phase of the emulsion; admixingthe aqueous solution and oil phase to a smooth blend avoiding foamformation; emulsifying the resulting mixture; and printing theemulsified membrane components onto an electrode carrier.
 11. The methodof claim 10, wherein the emulsified membrane components are applied tothe electrode carrier by one of pin transfer printing and microdispensing.
 12. The method of claim 10, wherein the components of thehydrophilic compartment include a hydrophilic binder polymer, anemulsifier and a salt.
 13. The method of claim 10, wherein the oil phaseof the emulsion includes a hydrophobic polymer.
 14. The method of claim13, wherein the oil phase further includes a cross-linker.
 15. Themethod of claim 10, wherein the step of emulsifying is carried out onice and the shear rate during the emulsifying is gradually increaseduntil a specific surface area of about 2.5 m²/mL is achieved,corresponding to a mean particle dimension of less than 1 micrometer.16. An electrochemical sensing device for the analysis of an aqueoussample, comprising a diagnostic card body, and an electrode as definedin claim 2 mounted to the card body.
 17. The electrochemical sensingdevice as defined in claim 16, for use as a potentiometric referenceelectrode, wherein one of the hydrophilic compartment and thehydrophilic layer contains bicarbonate salt and a pH sensitive redoxsalt.
 18. The electrochemical sensing device as defined in claim 17,wherein the hydrophilic compartment comprises a salt composition whichis approximately equi-transferrent.
 19. The electrochemical sensingdevice as defined in claim 16, wherein the hydrophobic compartmentcontains acrylated siloxane and the hydrophilic compartment containspolyvinylalcohol.
 20. The electrochemical sensing device of claim 16,for use as a polarographic oxygen sensor, wherein the heterogeneousmembrane has an oxygen permeability of less than 6×10⁻¹³ mole cm⁻¹ s⁻¹atm⁻¹.
 21. The electrochemical sensing device of claim 20, wherein thehydrophobic compartment contains epoxy-polyol and the hydrophiliccompartment contains polyvinylalcohol.
 22. The electrochemical device ofclaim 17, wherein the one of the hydrophilic compartment and hydrophiliclayer further contains carbonic anhydrase.